Cardio-respirometry disruption in zebrafish (Danio rerio) embryos exposed to hydraulic fracturing flowback and produced water.

PubMed

Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

2017-12-01

Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24-72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO 2 ), while for 5% FPW, both fractions reduced MO 2 . Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO 2 . Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Zebrafish (Danio rerio): A Potential Model for Toxinological Studies.

PubMed

Vargas, Rafael Antonio; Sarmiento, Karen; Vásquez, Isabel Cristina

2015-10-01

Zebrafish are an emerging basic biomedical research model that has multiple advantages compared with other research models. Given that biotoxins, such as toxins, poisons, and venoms, represent health hazards to animals and humans, a low-cost biological model that is highly sensitive to biotoxins is useful to understand the damage caused by such agents and to develop biological tests to prevent and reduce the risk of poisoning in potential cases of bioterrorism or food contamination. In this article, a narrative review of the general aspects of zebrafish as a model in basic biomedical research and various studies in the field of toxinology that have used zebrafish as a biological model are presented. This information will provide useful material to beginner students and researchers who are interested in developing toxinological studies with the zebrafish model.

The zebrafish as a model for complex tissue regeneration

PubMed Central

Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

2013-01-01

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

Polysaccharides from astragali radix restore chemical-induced blood vessel loss in zebrafish

PubMed Central

2012-01-01

Background Astragali Radix has been used widely for the treatment of cardiovascular and cerebrovascular diseases, and to enhance endurance and stamina in traditional Chinese medicine (TCM) for over 2000 years. The polysaccharide constituents of Astragali Radix (ARP) are considered as one of the major constituents contributing to the multiple pharmacological effects of this medicinal plant. The purpose of the study is to evaluate the vascular regenerative activities of ARPs in a chemically-induced blood vessel loss model in zebrafish. Methods Blood vessel loss was induced in both Tg(fli-1a:EGFP)y1 and Tg(fli-1a:nEGFP)y7 embryos by administration of 300 nM VEGFR tyrosine kinase inhibitor II (VRI) for 3 h at 24 hpf (hour post-fertilization). Then, the blood vessel damaged zebrafish were treated with ARPs for 21 h and 45 h after VRI withdrawal. Morphological changes in intersegmental vessels (ISVs) of zebrafish larvae were observed under the fluorescence microscope and measured quantitatively. The rescue effect of ARPs in the zebrafish models was validated by measuring the relative mRNA expressions of Kdrl, Kdr and Flt-1 using real-time PCR. Results Two polysaccharide fractions, P4 (50000 D < molecular weight & diameter < 0.1 μm) and P5 (molecular diameter > 0.1 μm), isolated from Astragali Radix by ultrafiltration, produced a significant and dose-dependent recovery in VRI-induced blood vessel loss in zebrafish. Furthermore, the down-regulation of Flk-1 and Flt-1 mRNA expression induced by VRI was reversed by treatment with P4. Conclusion The present study demonstrates that P4 isolated from Astragali Radix reduces VRI-induced blood vessel loss in zebrafish. These findings support the hypothesis that polysaccharides are one of the active constituents in Astragali Radix, contributing to its beneficial effect on treatment of diseases associated with a deficiency in angiogenesis. PMID:22357377

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

The zebrafish eye—a paradigm for investigating human ocular genetics

PubMed Central

Richardson, R; Tracey-White, D; Webster, A; Moosajee, M

2017-01-01

Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future. PMID:27612182

Zebrafish (Danio rerio) embryos as a model for testing proteratogens.

PubMed

Weigt, Stefan; Huebler, Nicole; Strecker, Ruben; Braunbeck, Thomas; Broschard, Thomas H

2011-03-15

Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B(1), carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene, which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish embryos exposed for 3 days. The test substances and/or the substance class produced characteristic patterns of fingerprint endpoints. Several substances produced effects that could be identified already at 1 dpf (days post fertilization), whereas the effects of others could only be identified unambiguously after hatching at ≥ 3 dpf. The LC₅₀ and EC₅₀ values were used to calculate the teratogenicity index (TI) for the different substances, and the EC₂₀ values were related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos are able to activate proteratogenic substances without addition of an exogenous metabolic activation system. Moreover, the teratogenic effects were observed at concentrations relevant to human exposure data. Along with other findings, our results indicate that zebrafish embryos are a useful alternative method for traditional teratogenicity testing with mammalian species. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The zebrafish world of colors and shapes: preference and discrimination.

PubMed

Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

2015-04-01

Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.

Quantum Dot Nanotoxicity Assessment Using the Zebrafish Embryo

PubMed Central

King-Heiden, Tisha C.; Wiecinski, Paige N.; Mangham, Andrew N.; Metz, Kevin M.; Nesbit, Dorothy; Pedersen, Joel A.; Hamers, Robert J.; Heideman, Warren; Peterson, Richard E.

2009-01-01

Quantum dots (QDs) hold promise for several biomedical, life sciences and photovoltaic applications. Substantial production volumes and environmental release are anticipated. QD toxicity may be intrinsic to their physicochemical properties, or result from the release of toxic components during breakdown. We hypothesized that developing zebrafish could be used to identify and distinguish these different types of toxicity. Embryos were exposed to aqueous suspensions of CdSecore/ZnSshell QDs functionalized with either poly-L-lysine or poly(ethylene glycol) terminated with methoxy, carboxylate, or amine groups. Toxicity was influenced by the QD coating, which also contributed to the QD suspension stability. At sublethal concentrations, many QD preparations produced characteristic signs of Cd toxicity that weakly correlated with metallothionein expression, indicating that QDs are only slightly degraded in vivo. QDs also produced distinctly different toxicity that could not be explained by Cd release. Using the zebrafish model, we were able to distinguish toxicity intrinsic to QDs from that caused by released metal ions. We conclude that developing zebrafish provide a rapid, low- cost approach for assessing structure-toxicity relationships of nanoparticles. PMID:19350942

[Construction and expression analysis of the zebrafish heart-specific transgenetic vector based on Tol2 transposable element].

PubMed

Chen, Tingfang; Luo, Na; Xie, Huaping; Wu, Xiushan; Deng, Yun

2010-02-01

In an effort to generate a desired expression construct for making heart-specific expression transgenic zebrafish, a Tol2 plasmid, which can drive EGFP reporter gene specifically expressed in the heart, was modified using subcloning technology. An IRES fragment bearing multiple cloning site (MCS) was amplified directly from pIRES2-EGFP plasmid and was inserted between the CMLC2 promoter and EGFP fragment of the pDestTol2CG vector. This recombinant expression plasmid pTol2-CMLC2-IRES-EGFP can drive any interested gene specifically expressed in the zebrafish heart along with EGFP reporter gene. To test the effectiveness of this new expression plasmid, we constructed pTol2-CMLC2-RED-IRES-EGFP plasmid by inserting another reporter gene DsRed-Monome into MCS downstream of the CMLC2 promoter and injected this transgenic recombinant plasmid into one-cell stage embryos of zebrafish. Under fluorescence microscope, both the red fluorescence and the green fluorescence produced by pTol2-CMLC2-RED-IRES-EGFP were detected specifically in the heart tissue in the same expression pattern. This novel expression construct pTol2-CMLC2-IRES-EGFP will become an important tool for our research on identifying heart development candidate genes' function using zebrafish as a model.

Functional and Structural Characterization of Zebrafish ASC.

PubMed

Li, Yajuan; Huang, Yi; Cao, Xiaocong; Yin, Xueying; Jin, Xiangyu; Liu, Sheng; Jiang, Jiansheng; Jiang, Wei; Xiao, Tsan Sam; Zhou, Rongbin; Cai, Gang; Hu, Bing; Jin, Tengchuan

2018-05-23

The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)) is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein (MBP). Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

PubMed

Okuda, Kazuhide Shaun; Tan, Pei Jean; Patel, Vyomesh

2016-04-01

Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

Zebrafish as a disease model for studying human hepatocellular carcinoma.

PubMed

Lu, Jeng-Wei; Ho, Yi-Jung; Yang, Yi-Ju; Liao, Heng-An; Ciou, Shih-Ci; Lin, Liang-In; Ou, Da-Liang

2015-11-14

Liver cancer is one of the world's most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury.

Zebrafish as a disease model for studying human hepatocellular carcinoma

PubMed Central

Lu, Jeng-Wei; Ho, Yi-Jung; Yang, Yi-Ju; Liao, Heng-An; Ciou, Shih-Ci; Lin, Liang-In; Ou, Da-Liang

2015-01-01

Liver cancer is one of the world’s most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury. PMID:26576090

Circadian rhythmicity and light sensitivity of the zebrafish brain.

PubMed

Moore, Helen A; Whitmore, David

2014-01-01

Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

Circadian Rhythmicity and Light Sensitivity of the Zebrafish Brain

PubMed Central

Moore, Helen A.; Whitmore, David

2014-01-01

Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Fan; Qin, Wei; Zhang, Jing-Pu; Hu, Chang-Qin

2015-01-01

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.

Anxiolytic-like effects of noribogaine in zebrafish.

PubMed

Kalueff, Allan V; Kaluyeva, Aleksandra; Maillet, Emeline L

2017-07-14

Noribogaine is the main psychoactive metabolite of the hallucinogenic drug ibogaine, and is a particularly interesting compound potentially useful to treat dependence and various psychiatric disorders. Here, we report the effects of noribogaine on anxiety and locomotion in zebrafish (Danio rerio), a new promising model organism in neurobehavioral and psychopharmacological research. Adult zebrafish were subjected to the 5min novel tank test (NTT) following an acute, 20-min drug immersion in 1, 5 and 10mg/L noribogaine. Overall, noribogaine produced robust anxiolytic-like behavior in zebrafish (increasing the time spent and transitions to the top half compartment and reducing freezing bouts) without overt effects on fish locomotion. Taken together, these results indicate that noribogaine modulates the components of the acute stress response related to emotionality and anxiety behaviors, implicating this drug as a potentially useful non-sedative anxiolytic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Culturable Gut Microbiota Diversity in Zebrafish

PubMed Central

Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-01-01

Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid

Culturable gut microbiota diversity in zebrafish.

PubMed

Cantas, Leon; Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

2012-03-01

The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A-D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional

Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.

PubMed

Egan, Rupert J; Bergner, Carisa L; Hart, Peter C; Cachat, Jonathan M; Canavello, Peter R; Elegante, Marco F; Elkhayat, Salem I; Bartels, Brett K; Tien, Anna K; Tien, David H; Mohnot, Sopan; Beeson, Esther; Glasgow, Eric; Amri, Hakima; Zukowska, Zofia; Kalueff, Allan V

2009-12-14

The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

PubMed

Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

2017-09-01

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Zebrafish embryology and cartilage staining protocols for high school students.

PubMed

Emran, Farida; Brooks, Jacqueline M; Zimmerman, Steven R; Johnson, Susan L; Lue, Robert A

2009-06-01

The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

PubMed Central

Appelbaum, Lior; Wang, Gordon X.; Maro, Geraldine S.; Mori, Rotem; Tovin, Adi; Marin, Wilfredo; Yokogawa, Tohei; Kawakami, Koichi; Smith, Stephen J.; Gothilf, Yoav; Mignot, Emmanuel; Mourrain, Philippe

2009-01-01

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation. PMID:19966231

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Characterization of synergistic embryotoxicity of nickel and buprofezin in zebrafish.

PubMed

Ku, Tingting; Yan, Wei; Jia, Wuyao; Yun, Yang; Zhu, Na; Li, Guangke; Sang, Nan

2015-04-07

Multiple pollutants, usually at low levels, coexist and may interact in the environment. It is therefore important to analyze the toxicity of mixtures of coexisting chemicals to evaluate the potential ecological risk. Concern regarding the co-occurrence and combined bioeffects of heavy metals and organic insecticides in aquatic settings has existed for many years, but a clear understanding of the interactions between and potential combined toxicity of these chemicals remains elusive. In the present study, the combined effects of the heavy metal nickel (NiSO4) and insect growth regulator buprofezin on the induction of embryo toxicity in zebrafish were assessed. By applying nonlinear regression to the concentration-response data with each of the chemicals using the Hill and Langmuir functions and computing the predictions using the model of concentration addition (CA), we confirmed that NiSO4 and buprofezin acted together to produce synergistic embryotoxicity in zebrafish. Subsequently, we further found that the combination of NiSO4 and buprofezin formed a complex that facilitated the uptake of nickel (Ni) and buprofezin by the embryos. Following this, we clarified that an oxidative mechanism of the complex might underlie the synergistic embryotoxicity of NiSO4 and buprofezin.

Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

PubMed

Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

2014-08-01

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

A Novel Method for Rearing Zebrafish by Using Freshwater Rotifers (Brachionus calyciflorus)

PubMed Central

Aoyama, Yuta; Moriya, Natsumi; Tanaka, Shingo; Taniguchi, Tomoko; Hosokawa, Hiroshi

2015-01-01

Abstract The zebrafish (Danio rerio) has become a powerful model organism for studying developmental processes and genetic diseases. However, there remain several problems in previous rearing methods. In this study, we demonstrate a novel method for rearing zebrafish larvae by using a new first food, freshwater rotifers (Brachionus calyciflorus). Feeding experiments indicated that freshwater rotifers are suitable as the first food for newly hatched larval fish. In addition, we revisited and improved a feeding schedule from 5 to 40 days postfertilization. Our feeding method using freshwater rotifers accelerated larval growth. At 49 dpf, one pair out of 10 pairs successfully produced six fertilized eggs. At 56, 63, and 71 dpf, 6 out of the 10 pairs constantly produced normal embryos. Our method will improve the husbandry of the zebrafish. PMID:25938499

Developmental Exposure to a Dopaminergic Toxicant Produces Altered Locomotor Activity in Larval Zebrafish

EPA Science Inventory

In an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we are characterizing the locomotor activity of zebrafish (Danio rerio) larvae after developmental exposure to various classes of prototypic drugs that act on the central nervous system. ...

Multiple Sex-Associated Regions and a Putative Sex Chromosome in Zebrafish Revealed by RAD Mapping and Population Genomics

PubMed Central

Anderson, Jennifer L.; Rodríguez Marí, Adriana; Braasch, Ingo; Amores, Angel; Hohenlohe, Paul; Batzel, Peter; Postlethwait, John H.

2012-01-01

Within vertebrates, major sex determining genes can differ among taxa and even within species. In zebrafish (Danio rerio), neither heteromorphic sex chromosomes nor single sex determination genes of large effect, like Sry in mammals, have yet been identified. Furthermore, environmental factors can influence zebrafish sex determination. Although progress has been made in understanding zebrafish gonad differentiation (e.g. the influence of germ cells on gonad fate), the primary genetic basis of zebrafish sex determination remains poorly understood. To identify genetic loci associated with sex, we analyzed F2 offspring of reciprocal crosses between Oregon *AB and Nadia (NA) wild-type zebrafish stocks. Genome-wide linkage analysis, using more than 5,000 sequence-based polymorphic restriction site associated (RAD-tag) markers and population genomic analysis of more than 30,000 single nucleotide polymorphisms in our *ABxNA crosses revealed a sex-associated locus on the end of the long arm of chr-4 for both cross families, and an additional locus in the middle of chr-3 in one cross family. Additional sequencing showed that two SNPs in dmrt1 previously suggested to be functional candidates for sex determination in a cross of ABxIndia wild-type zebrafish, are not associated with sex in our AB fish. Our data show that sex determination in zebrafish is polygenic and that different genes may influence sex determination in different strains or that different genes become more important under different environmental conditions. The association of the end of chr-4 with sex is remarkable because, unique in the karyotype, this chromosome arm shares features with known sex chromosomes: it is highly heterochromatic, repetitive, late replicating, and has reduced recombination. Our results reveal that chr-4 has functional and structural properties expected of a sex chromosome. PMID:22792396

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development

PubMed Central

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W.; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2015-01-01

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. PMID:26427652

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

PubMed

Ichijo, Yuta; Mochimaru, Yuta; Azuma, Morio; Satou, Kazuhiro; Negishi, Jun; Nakakura, Takashi; Oshima, Natsuki; Mogi, Chihiro; Sato, Koichi; Matsuda, Kouhei; Okajima, Fumikazu; Tomura, Hideaki

2016-01-01

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. Copyright © 2015 Elsevier Inc. All rights reserved.

An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio)

PubMed Central

Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

2014-01-01

The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterize ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 minutes of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 minutes following withdrawal from chronic ethanol exposure (0.50% v/v). We characterize the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on 3 domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish. PMID:24598276

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Recording Field Potentials From Zebrafish Larvae During Escape Responses

PubMed Central

Monesson-Olson, Bryan D.; Troconis, Eileen L.; Trapani, Josef G.

2014-01-01

Among vertebrates, startle responses are a ubiquitous method for alerting, and avoiding or escaping from alarming or dangerous stimuli. In zebrafish larvae, fast escape behavior is easily evoked through either acoustic or tactile stimuli. For example, a light touch to the head will excite trigeminal neurons that in turn excite a large reticulospinal neuron in the hindbrain called the Mauthner cell (M-cell). The M-cell action potential then travels down the contralateral trunk of the larva exciting motoneurons, which subsequently excite the entire axial musculature, producing a large amplitude body bend away from the source of the stimulus. This body conformation is known as the “C-bend” due to the shape of the larva during the behavior. As a result of the semi-synchronized activation of the M-cell, the population of motor neurons, and the axial trunk muscles, a large field potential is generated and can be recorded from free-swimming or fixed-position larvae. Undergraduate laboratories that record field potentials during escape responses in larval zebrafish are relatively simple to setup and allow students to observe and study the escape reflex circuit. Furthermore, by testing hypotheses, analyzing data and writing journal-style laboratory reports, students have multiple opportunities to learn about many neuroscience topics including vertebrate reflexes; sensory transduction; synaptic-, neuro-, and muscle-physiology; the M-cell mediated escape response; and the zebrafish as a model organism. Here, we detail the equipment, software, and recording setup necessary to observe field potentials in an undergraduate teaching lab. Additionally, we discuss potential advanced laboratory exercises and pedagogical outcomes. Finally, we note possible low-cost alternatives for recording field potentials. PMID:25565920

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.

PubMed

Buckles, Gerri R; Thorpe, Christopher J; Ramel, Marie-Christine; Lekven, Arne C

2004-05-01

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.

Repairing quite swimmingly: advances in regenerative medicine using zebrafish.

PubMed

Goessling, Wolfram; North, Trista E

2014-07-01

Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future. © 2014. Published by The Company of Biologists Ltd.

Effect of acute ethanol administration on zebrafish tail-beat motion.

PubMed

Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2015-11-01

Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening. Copyright © 2015 Elsevier Inc. All rights reserved.

Advances in the Study of Heart Development and Disease Using Zebrafish

PubMed Central

Brown, Daniel R.; Samsa, Leigh Ann; Qian, Li; Liu, Jiandong

2016-01-01

Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications. PMID:27335817

Influence of robotic shoal size, configuration, and activity on zebrafish behavior in a free-swimming environment.

PubMed

Butail, Sachit; Polverino, Giovanni; Phamduy, Paul; Del Sette, Fausto; Porfiri, Maurizio

2014-12-15

In animal studies, robots have been recently used as a valid tool for testing a wide spectrum of hypotheses. These robots often exploit visual or auditory cues to modulate animal behavior. The propensity of zebrafish, a model organism in biological studies, toward fish with similar color patterns and shape has been leveraged to design biologically inspired robots that successfully attract zebrafish in preference tests. With an aim of extending the application of such robots to field studies, here, we investigate the response of zebrafish to multiple robotic fish swimming at different speeds and in varying arrangements. A soft real-time multi-target tracking and control system remotely steers the robots in circular trajectories during the experimental trials. Our findings indicate a complex behavioral response of zebrafish to biologically inspired robots. More robots produce a significant change in salient measures of stress, with a fast robot swimming alone causing more freezing and erratic activity than two robots swimming slowly together. In addition, fish spend more time in the proximity of a robot when they swim far apart than when the robots swim close to each other. Increase in the number of robots also significantly alters the degree of alignment of fish motion with a robot. Results from this study are expected to advance our understanding of robot perception by live animals and aid in hypothesis-driven studies in unconstrained free-swimming environments. Copyright © 2014 Elsevier B.V. All rights reserved.

Episodic swimming in the larval zebrafish is generated by a spatially distributed spinal network with modular functional organization

PubMed Central

Wiggin, Timothy D.; Anderson, Tatiana M.; Eian, John; Peck, Jack H.

2012-01-01

Despite the diverse methods vertebrates use for locomotion, there is evidence that components of the locomotor central pattern generator (CPG) are conserved across species. When zebrafish begin swimming early in development, they perform short episodes of activity separated by periods of inactivity. Within these episodes, the trunk flexes with side-to-side alternation and the traveling body wave progresses rostrocaudally. To characterize the distribution of the swimming CPG along the rostrocaudal axis, we performed transections of the larval zebrafish spinal cord and induced fictive swimming using N-methyl-d-aspartate (NMDA). In both intact and spinalized larvae, bursting is found throughout the rostrocaudal extent of the spinal cord, and the properties of fictive swimming observed were dependent on the concentration of NMDA. We isolated series of contiguous spinal segments by performing multiple spinal transections on the same larvae. Although series from all regions of the spinal cord have the capacity to produce bursts, the capacity to produce organized episodes of fictive swimming has a rostral bias: in the rostral spinal cord, only 12 contiguous body segments are necessary, whereas 23 contiguous body segments are necessary in the caudal spinal cord. Shorter series of segments were often active but produced either continuous rhythmic bursting or sporadic, nonrhythmic bursting. Both episodic and continuous bursting alternated between the left and right sides of the body and showed rostrocaudal progression, demonstrating the functional dissociation of the circuits responsible for episodic structure and fine burst timing. These findings parallel results in mammalian locomotion, and we propose a hierarchical model of the larval zebrafish swimming CPG. PMID:22572943

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development.

PubMed

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric C; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2016-01-05

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Methods to study maternal regulation of germ cell specification in zebrafish

PubMed Central

Kaufman, O.H.; Marlow, F.L.

2016-01-01

The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine.

PubMed

Crim, Marcus J; Lawrence, Christian; Livingston, Robert S; Rakitin, Andrei; Hurley, Shane J; Riley, Lela K

2017-07-01

Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish.

Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine

PubMed Central

Crim, Marcus J; Lawrence, Christian; Livingston, Robert S; Rakitin, Andrei; Hurley, Shane J; Riley, Lela K

2017-01-01

Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish. PMID:28724491

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

PubMed

Lin, Heng-Ju; Lee, Shu-Hua; Wu, Jen-Leih; Duann, Yeh-Fang; Chen, Jyh-Yih

2013-12-01

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

PubMed

Winter, Matthew J; Windell, Dylan; Metz, Jeremy; Matthews, Peter; Pinion, Joe; Brown, Jonathan T; Hetheridge, Malcolm J; Ball, Jonathan S; Owen, Stewart F; Redfern, Will S; Moger, Julian; Randall, Andrew D; Tyler, Charles R

2017-07-26

Functional neuroimaging, using genetically-encoded Ca 2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

PubMed

Yin, L; Maddison, L A; Chen, W

2016-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Neurochemical measurements in the zebrafish brain

PubMed Central

Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

2015-01-01

The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

Diving deeper into Zebrafish development of social behavior: analyzing high resolution data.

PubMed

Buske, Christine; Gerlai, Robert

2014-08-30

Vertebrate model organisms have been utilized in high throughput screening but only with substantial cost and human capital investment. The zebrafish is a vertebrate model species that is a promising and cost effective candidate for efficient high throughput screening. Larval zebrafish have already been successfully employed in this regard (Lessman, 2011), but adult zebrafish also show great promise. High throughput screening requires the use of a large number of subjects and collection of substantial amount of data. Collection of data is only one of the demanding aspects of screening. However, in most screening approaches that involve behavioral data the main bottleneck that slows throughput is the time consuming aspect of analysis of the collected data. Some automated analytical tools do exist, but often they only work for one subject at a time, eliminating the possibility of fully utilizing zebrafish as a screening tool. This is a particularly important limitation for such complex phenotypes as social behavior. Testing multiple fish at a time can reveal complex social interactions but it may also allow the identification of outliers from a group of mutagenized or pharmacologically treated fish. Here, we describe a novel method using a custom software tool developed within our laboratory, which enables tracking multiple fish, in combination with a sophisticated analytical approach for summarizing and analyzing high resolution behavioral data. This paper focuses on the latter, the analytic tool, which we have developed using the R programming language and environment for statistical computing. We argue that combining sophisticated data collection methods with appropriate analytical tools will propel zebrafish into the future of neurobehavioral genetic research. Copyright © 2014. Published by Elsevier B.V.

Zebrafish as tools for drug discovery.

PubMed

MacRae, Calum A; Peterson, Randall T

2015-10-01

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Phylogeny of zebrafish, a "model species," within Danio, a "model genus".

PubMed

McCluskey, Braedan M; Postlethwait, John H

2015-03-01

Zebrafish (Danio rerio) is an important model for vertebrate development, genomics, physiology, behavior, toxicology, and disease. Additionally, work on numerous Danio species is elucidating evolutionary mechanisms for morphological development. Yet, the relationships of zebrafish and its closest relatives remain unclear possibly due to incomplete lineage sorting, speciation with gene flow, and interspecies hybridization. To clarify these relationships, we first constructed phylogenomic data sets from 30,801 restriction-associated DNA (RAD)-tag loci (483,026 variable positions) with clear orthology to a single location in the sequenced zebrafish genome. We then inferred a well-supported species tree for Danio and tested for gene flow during the diversification of the genus. An approach independent of the sequenced zebrafish genome verified all inferred relationships. Although identification of the sister taxon to zebrafish has been contentious, multiple RAD-tag data sets and several analytical methods provided strong evidence for Danio aesculapii as the most closely related extant zebrafish relative studied to date. Data also displayed patterns consistent with gene flow during speciation and postspeciation introgression in the lineage leading to zebrafish. The incorporation of biogeographic data with phylogenomic analyses put these relationships in a phylogeographic context and supplied additional support for D. aesculapii as the sister species to D. rerio. The clear resolution of this study establishes a framework for investigating the evolutionary biology of Danio and the heterogeneity of genome evolution in the recent history of a model organism within an emerging model genus for genetics, development, and evolution. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Simple, economical heat-shock devices for zebrafish housing racks.

PubMed

Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

2011-12-01

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae.

PubMed

Souder, Jaclyn Paige; Gorelick, Daniel A

2017-08-01

Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis

PubMed Central

Hayes, Anthony J; Mitchell, Ruth E; Bashford, Andrew; Reynolds, Scott; Caterson, Bruce; Hammond, Chrissy L

2013-01-01

Background: The zebrafish is an important developmental model. Surprisingly, there are few studies that describe the glycosaminoglycan composition of its extracellular matrix during skeletogenesis. Glycosaminoglycans on proteoglycans contribute to the material properties of musculo skeletal connective tissues, and are important in regulating signalling events during morphogenesis. Sulfation motifs within the chain structure of glycosaminoglycans on cell-associated and extracellular matrix proteoglycans allow them to bind and regulate the sequestration/presentation of bioactive signalling molecules important in musculo-skeletal development. Results: We describe the spatio-temporal expression of different glycosaminoglycan moieties during zebrafish skeletogenesis with antibodies recognising (1) native sulfation motifs within chondroitin and keratan sulfate chains, and (2) enzyme-generated neoepitope sequences within the chain structure of chondroitin sulfate (i.e., 0-, 4-, and 6-sulfated isoforms) and heparan sulfate glycosaminoglycans. We show that all the glycosaminoglycan moieties investigated are expressed within the developing skeletal tissues of larval zebrafish. However, subtle changes in their patterns of spatio-temporal expression over the period examined suggest that their expression is tightly and dynamically controlled during development. Conclusions: The subtle differences observed in the domains of expression between different glycosaminoglycan moieties suggest differences in their functional roles during establishment of the primitive analogues of the skeleton. Developmental Dynamics 242:778–789, 2013. © 2013 Wiley Periodicals, Inc. Key Findings The developing zebrafish skeleton expresses many different glycosaminoglycan modifications. Multiple different glycosaminoglycan epitopes are dynamically expressed in the craniofacial skeleton. Expression of chondroitin sulfate moieties are dynamically expressed in the vertebral column and precede

Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis.

PubMed

Liu, Ting Xi; Howlett, Niall G; Deng, Min; Langenau, David M; Hsu, Karl; Rhodes, Jennifer; Kanki, John P; D'Andrea, Alan D; Look, A Thomas

2003-12-01

Mechanisms underlying the multiple developmental defects observed in Fanconi anemia (FA) patients are not well defined. We have identified the zebrafish homolog of human FANCD2, which encodes a nuclear effector protein that is monoubiquitinated in response to DNA damage, targeting it to nuclear foci where it preserves chromosomal integrity. Fancd2-deficient zebrafish embryos develop defects similar to those found in children with FA, including shortened body length, microcephaly, and microophthalmia, which are due to extensive cellular apoptosis. Developmental defects and increased apoptosis in Fancd2-deficient zebrafish were corrected by injection of human FANCD2 or zebrafish bcl2 mRNA, or by knockdown of p53, indicating that in the absence of Fancd2, developing tissues spontaneously undergo p53-dependent apoptosis. Thus, Fancd2 is essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high levels of proliferative expansion, implicating this mechanism in the congenital abnormalities observed in human infants with FA.

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

PubMed

Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing; Yi, Meisheng; Hong, Yunhan

2012-08-10

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Interordinal Chimera Formation Between Medaka and Zebrafish for Analyzing Stem Cell Differentiation

PubMed Central

Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing

2012-01-01

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos. PMID:22204449

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly

PubMed Central

Sun, Qingrui; Liu, Xingfeng; Gong, Bo; Wu, Di; Meng, Anming; Jia, Shunji

2017-01-01

During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly. PMID:28924386

Zebrafish and Streptococcal Infections.

PubMed

Saralahti, A; Rämet, M

2015-09-01

Streptococcal bacteria are a versatile group of gram-positive bacteria capable of infecting several host organisms, including humans and fish. Streptococcal species are common colonizers of the human respiratory and gastrointestinal tract, but they also cause some of the most common life-threatening, invasive infections in humans and aquaculture. With its unique characteristics and efficient tools for genetic and imaging applications, the zebrafish (Danio rerio) has emerged as a powerful vertebrate model for infectious diseases. Several zebrafish models introduced so far have shown that zebrafish are suitable models for both zoonotic and human-specific infections. Recently, several zebrafish models mimicking human streptococcal infections have also been developed. These models show great potential in providing novel information about the pathogenic mechanisms and host responses associated with human streptococcal infections. Here, we review the zebrafish infection models for the most relevant streptococcal species: the human-specific Streptococcus pneumoniae and Streptococcus pyogenes, and the zoonotic Streptococcus iniae and Streptococcus agalactiae. The recent success and the future potential of these models for the study of host-pathogen interactions in streptococcal infections are also discussed. © 2015 The Foundation for the Scandinavian Journal of Immunology.

Measuring zebrafish turning rate.

PubMed

Mwaffo, Violet; Butail, Sachit; di Bernardo, Mario; Porfiri, Maurizio

2015-06-01

Zebrafish is becoming a popular animal model in preclinical research, and zebrafish turning rate has been proposed for the analysis of activity in several domains. The turning rate is often estimated from the trajectory of the fish centroid that is output by commercial or custom-made target tracking software run on overhead videos of fish swimming. However, the accuracy of such indirect methods with respect to the turning rate associated with changes in heading during zebrafish locomotion is largely untested. Here, we compare two indirect methods for the turning rate estimation using the centroid velocity or position data, with full shape tracking for three different video sampling rates. We use tracking data from the overhead video recorded at 60, 30, and 15 frames per second of zebrafish swimming in a shallow water tank. Statistical comparisons of absolute turning rate across methods and sampling rates indicate that, while indirect methods are indistinguishable from full shape tracking, the video sampling rate significantly influences the turning rate measurement. The results of this study can aid in the selection of the video capture frame rate, an experimental design parameter in zebrafish behavioral experiments where activity is an important measure.

INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

EPA Science Inventory

To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

PubMed Central

Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

2013-01-01

Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

Effects of abnormal light-rearing conditions on retinal physiology in larvae zebrafish.

PubMed

Saszik, S; Bilotta, J

1999-11-01

Anatomic studies have found that zebrafish retinal neurons develop in a sequential fashion. In addition, exposure to abnormal light-rearing conditions produces deficits in visual behavior of larvae zebrafish, even though there appears to be little effect of the light-rearing conditions on the gross morphology of the retina. The purpose of this study was to assess the effects of abnormal light-rearing conditions on larvae zebrafish retinal physiology. Larvae zebrafish (Danio rerio) were exposed to constant light (LL), constant dark (DD), or normal cyclic light (LD) from fertilization to 6 days postfertilization (dpf). After 6 days, the animals were placed into normal cyclic light and tested at 6 to 8, 13 to 15, and 21 to 24 dpf. Electroretinogram (ERG) responses to visual stimuli, consisting of various wavelengths and irradiances, were recorded. Comparisons were made across the three age groups and the three light-rearing conditions. Deficits from the light-rearing conditions were seen immediately after exposure (6 8 dpf). The LL-condition subjects showed the greatest deficit in the UV and short-wavelength areas and the DD-condition subjects showed a slight deficit across the entire spectrum. At 13 to 15 dpf, the LL and DD groups showed an increase in sensitivity and by 21 to 24 dpf, the groups no longer differed from controls. Abnormal lighting environments can adversely influence the physiological development of the larvae zebrafish retina. The pattern of damage that was seen in zebrafish is similar to that found in other vertebrates, including higher vertebrates. However, unlike higher vertebrates, the zebrafish appears to be capable of regeneration. This suggests that the zebrafish would be a viable model for light environment effects and neural regeneration.

Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism

PubMed Central

Zhao, Lu; Xia, Zhidan; Wang, Fudi

2014-01-01

Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases. PMID:24639652

Zebrafish Embryo Toxicity Microscale Model for Ichthyotoxicity Evaluation of Marine Natural Products.

PubMed

Bai, Hong; Kong, Wen-Wen; Shao, Chang-Lun; Li, Yun; Liu, Yun-Zhang; Liu, Min; Guan, Fei-Fei; Wang, Chang-Yun

2016-04-01

Marine organisms often protect themselves against their predators by chemical defensive strategy. The second metabolites isolated from marine organisms and their symbiotic microbes have been proven to play a vital role in marine chemical ecology, such as ichthyotoxicity, allelopathy, and antifouling. It is well known that the microscale models for marine chemoecology assessment are urgently needed for trace quantity of marine natural products. Zebrafish model has been widely used as a microscale model in the fields of environment ecological evaluation and drug safety evaluation, but seldom reported for marine chemoecology assessment. In this work, zebrafish embryo toxicity microscale model was established for ichthyotoxicity evaluation of marine natural products by using 24-well microplate based on zebrafish embryo. Ichthyotoxicity was evaluated by observation of multiple toxicological endpoints, including coagulation egg, death, abnormal heartbeat, no spontaneous movement, delayed hatch, and malformation of the different organs during zebrafish embryogenesis periods at 24, 48, and 72 h post-fertilization (hpf). 3,4-Dichloroaniline was used as the positive control for method validation. Subsequently, the established model was applied to test the ichthyotoxic activity of the compounds isolated from corals and their symbiotic microbes and to isolate the bioactive secondary metabolites from the gorgonian Subergorgia mollis under bioassay guidance. It was suggested that zebrafish embryo toxicity microscale model is suitable for bioassay-guided isolation and preliminary bioactivity screening of marine natural products.

Intrinsic Properties of Larval Zebrafish Neurons in Ethanol

PubMed Central

Ikeda, Hiromi; Delargy, Alison H.; Yokogawa, Tohei; Urban, Jason M.; Burgess, Harold A.; Ono, Fumihito

2013-01-01

The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca2+] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets. PMID:23658822

Maintenance of Zebrafish Lines at the European Zebrafish Resource Center.

PubMed

Geisler, Robert; Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

2016-07-01

We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.

Toxic effects of brominated indoles and phenols on zebrafish embryos.

PubMed

Kammann, U; Vobach, M; Wosniok, W

2006-07-01

Organobromine compounds in the marine environment have been the focus of growing attention in past years. In contrast to anthropogenic brominated flame retardants, other brominated compounds are produced naturally, e.g., by common polychaete worms and algae. Brominated phenols and indoles assumed to be of biogenic origin have been detected in water and sediment extracts from the German Bight. These substances as well as some of their isomers have been tested with the zebrafish embryo test and were found to cause lethal as well as nonlethal malformations. The zebrafish test was able to detect a log K(OW)-related toxicity for bromophenols, suggesting nonpolar narcosis as a major mode of action. Different effect patterns could be observed for brominated indoles and bromophenols. The comparison of effective concentrations in the zebrafish embryo test with the concentrations determined in water samples suggests the possibility that brominated indoles may affect early life stages of marine fish species in the North Sea.

Effects of piracetam on behavior and memory in adult zebrafish.

PubMed

Grossman, Leah; Stewart, Adam; Gaikwad, Siddharth; Utterback, Eli; Wu, Nadine; Dileo, John; Frank, Kevin; Hart, Peter; Howard, Harry; Kalueff, Allan V

2011-04-25

Piracetam, a derivative of γ-aminobutyric acid, exerts memory-enhancing and mild anxiolytic effects in human and rodent studies. To examine the drug's behavioral profile further, we assessed its effects on behavioral and endocrine (cortisol) responses of adult zebrafish (Danio rerio)--a novel model species rapidly gaining popularity in neurobehavioral research. Overall, acute piracetam did not affect zebrafish novel tank and light-dark box behavior at mild doses (25-400mg/L), but produced nonspecific behavioral inhibition at 700mg/L. No effects on cortisol levels or inter-/intra-session habituation in the novel tank test were observed for acute or chronic mild non-sedative dose of 200mg/L. In contrast, fish exposed to chronic piracetam at this dose performed significantly better in the cued learning plus-maze test. This observation parallels clinical and rodent literature on the behavioral profile of piracetam, supporting the utility of zebrafish paradigms for testing nootropic agents. Copyright © 2011 Elsevier Inc. All rights reserved.

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.

PubMed

Hirsinger, Estelle; Steventon, Ben

2017-01-26

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Biological response of zebrafish after short-term exposure to azoxystrobin.

PubMed

Jiang, Jinhua; Shi, Yan; Yu, Ruixian; Chen, Liping; Zhao, Xueping

2018-07-01

Azoxystrobin (AZ) is a broad-spectrum systemic fungicide that widely used in the world. The present study investigated the toxicity effects on zebrafish after short-term exposure of AZ. Results demonstrated that the larval stage was most susceptible to AZ in the multiple life stages of zebrafish, with 96 h-LC 50 value of 0.777 mg/L. Zebrafish larvae were exposed to different AZ concentrations (0, 0.1, 1, 10, 100 μg/L) and examined on 24, 48 and 72 h. It was found that AZ induced ROS accumulation, increased GST, GPX and POD activity and the transcriptions of antioxidant and stress response related genes, while the opposite trend occurred for SOD and CAT activity in 24-h or 48-h exposure period. The increased E 2 and VTG levels in zebrafish larvae, and altered transcription levels of regulatory and steroidogenic genes in the hypothalamus-pituitary-gonad (HPG) axis indicated the endocrine disruption capacity of AZ. The transcripts of mdm2, p53, ogg1, bcl2, bbc3, cas8 and cas9 involved in cell apoptosis, and the mRNA levels of cytokines and chemokines such as cxcl-c1c, ccl, il-1β, il-8, ifn, and tnfα were in accordance with the trends of the examined genes involved in oxidative stress and endocrine system. The results suggested that short-term exposure to AZ might impose ecotoxicological effects on zebrafish larvae, and the information presented here also provide molecular strategies and increase mechanistic understanding of AZ-induced toxic response, and help elucidate the environmental risks of AZ. Copyright © 2018. Published by Elsevier Ltd.

Viral Diseases in Zebrafish: What Is Known and Unknown

PubMed Central

Crim, Marcus J.; Riley, Lela K.

2013-01-01

Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges

PubMed Central

Riesco, Marta F.; Robles, Vanesa

2013-01-01

Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs) containing primordial germ cells (PGCs) considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (cxcr4b, pou5f1, vasa and sox2) and upregulation of heat shock proteins (hsp70, hsp90). The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (eIF2S1, and LHCGR). Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs). Finally, we employed real-time PCR (qPCR) technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique. PMID:23805321

simUfish: An Interactive Application to Teach K-12 Students About Zebrafish Behavior.

PubMed

Mwaffo, Violet; Korneyeva, Veronika; Porfiri, Maurizio

2017-10-01

As the zebrafish is rapidly becoming a species of choice in preclinical research, several efforts are being placed toward creating educational programs for K-12 students based on this promising model organism. However, as any other model organisms, the use of zebrafish in classroom settings requires additional experimental resources and poses ethical challenges related to animal use. To mitigate these factors, we have developed an application (app), simUfish, which implements a mathematical model of zebrafish behavior for generating multiple fish trajectories and animating their body undulations. simUfish is developed using a multiplatform game engine and is expected to promote the knowledge of zebrafish behavior to both K-12 students and the general public. Specifically, it demonstrates basic principles of fish individual and social behaviors, including environment interaction; fear response toward a predator; shoaling; and attraction toward a stimulus, which can be a food source or simply a finger placed on the touch screen. The effectiveness of the app as an accessible experimental tool for learning was tested in an outreach activity on middle school students from the New York City school system. The results from this activity show an immediate, tangible improvement of students' satisfaction and willingness to learn about key concepts on zebrafish behavior, accompanied by high level of interest in life sciences.

OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.

PubMed

Akagi, Jin; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

2014-01-02

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Copyright © 2014 John Wiley & Sons, Inc.

Automated Planar Tracking the Waving Bodies of Multiple Zebrafish Swimming in Shallow Water.

PubMed

Wang, Shuo Hong; Cheng, Xi En; Qian, Zhi-Ming; Liu, Ye; Chen, Yan Qiu

2016-01-01

Zebrafish (Danio rerio) is one of the most widely used model organisms in collective behavior research. Multi-object tracking with high speed camera is currently the most feasible way to accurately measure their motion states for quantitative study of their collective behavior. However, due to difficulties such as their similar appearance, complex body deformation and frequent occlusions, it is a big challenge for an automated system to be able to reliably track the body geometry of each individual fish. To accomplish this task, we propose a novel fish body model that uses a chain of rectangles to represent fish body. Then in detection stage, the point of maximum curvature along fish boundary is detected and set as fish nose point. Afterwards, in tracking stage, we firstly apply Kalman filter to track fish head, then use rectangle chain fitting to fit fish body, which at the same time further judge the head tracking results and remove the incorrect ones. At last, a tracklets relinking stage further solves trajectory fragmentation due to occlusion. Experiment results show that the proposed tracking system can track a group of zebrafish with their body geometry accurately even when occlusion occurs from time to time.

Zebrafish Melanoma.

PubMed

Kaufman, Charles K

2016-01-01

Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology.

Alterations to Juvenile Zebrafish (Danio rerio) Swim Performance after Acute Embryonic Exposure to Sub-lethal Exposures of Hydraulic Fracturing Flowback and Produced Water.

PubMed

Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

2017-12-01

Hydraulic fracturing flowback and produced water (FPW) is a wastewater produced during fracturing activities in an operating well which is hyper saline and chemically heterogeneous in nature, containing both anthropogenic and petrogenic chemicals. Determination of FPW associated toxicity to embryonic fish is limited, while investigation into how embryonic exposures may affect later life stages is not yet studied. Zebrafish embryos (24hrs post fertilization) were acutely exposed to 2.5% and 5% FPW fractions for either 24 or 48hrs and returned to freshwater. After either 24 or 48h exposures, embryos were examined for expression of 3 hypoxia related genes. Erythropoietin (epoa) but not hypoxia inducible factor (hif1aa) nor hemoglobin -ß chain (hbbe1.1) was up-regulated after either 24 or 48h FPW exposure. Surviving embryos were placed in freshwater and grown to a juvenile stage (60days post fertilization). Previously exposed zebrafish were analyzed for both swim performance (U crit and U max ) and aerobic capacity. Fish exposed to both sediment containing (FPW-S) or sediment free (FPW-SF) FPW displayed significantly reduced aerobic scope and U crit /U max values compared to control conditions. Our results collectively suggest that organics present in our FPW sample may be responsible for sub-lethal fitness and metabolic responses. We provide evidence supporting the theory that the cardio-respiratory system is impacted by FPW exposure. This is the first known research associating embryonic FPW exposures to sub-lethal performance related responses in later life fish stages. Copyright © 2017 Elsevier B.V. All rights reserved.

What is the Thalamus in Zebrafish?

PubMed Central

Mueller, Thomas

2012-01-01

Current research on the thalamus and related structures in the zebrafish diencephalon identifies an increasing number of both neurological structures and ontogenetic processes as evolutionary conserved between teleosts and mammals. The patterning processes, for example, which during the embryonic development of zebrafish form the thalamus proper appear largely conserved. Yet also striking differences between zebrafish and other vertebrates have been observed, particularly when we look at mature and histologically differentiated brains. A case in point is the migrated preglomerular complex of zebrafish which evolved only within the lineage of ray-finned fish and has no counterpart in mammals or tetrapod vertebrates. Based on its function as a sensory relay station with projections to pallial zones, the preglomerular complex has been compared to specific thalamic nuclei in mammals. However, no thalamic projections to the zebrafish dorsal pallium, which corresponds topologically to the mammalian isocortex, have been identified. Merely one teleostean thalamic nucleus proper, the auditory nucleus, projects to a part of the dorsal telencephalon, the pallial amygdala. Studies on patterning mechanisms identify a rostral and caudal domain in the embryonic thalamus proper. In both, teleosts and mammals, the rostral domain gives rise to GABAergic neurons, whereas glutamatergic neurons originate in the caudal domain of the zebrafish thalamus. The distribution of GABAergic derivatives in the adult zebrafish brain, furthermore, revealed previously overlooked thalamic nuclei and redefined already established ones. These findings require some reconsideration regarding the topological origin of these adult structures. In what follows, I discuss how evolutionary conserved and newly acquired features of the developing and adult zebrafish thalamus can be compared to the mammalian situation. PMID:22586363

Application of Zebrafish Model to Environmental Toxicology.

PubMed

Komoike, Yuta; Matsuoka, Masato

2016-01-01

Recently, a tropical freshwater fish, the zebrafish, has been generally used as a useful model organism in various fields of life science worldwide. The zebrafish model has also been applied to environmental toxicology; however, in Japan, it has not yet become widely used. In this review, we will introduce the biological and historical backgrounds of zebrafish as an animal model and their breeding. We then present the current status of toxicological experiments using zebrafish that were treated with some important environmental contaminants, including cadmium, organic mercury, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and tributyltin. Finally, the future possible application of genetically modified zebrafish to the study of environmental toxicology is discussed.

Detection of blob objects in microscopic zebrafish images based on gradient vector diffusion.

PubMed

Li, Gang; Liu, Tianming; Nie, Jingxin; Guo, Lei; Malicki, Jarema; Mara, Andrew; Holley, Scott A; Xia, Weiming; Wong, Stephen T C

2007-10-01

The zebrafish has become an important vertebrate animal model for the study of developmental biology, functional genomics, and disease mechanisms. It is also being used for drug discovery. Computerized detection of blob objects has been one of the important tasks in quantitative phenotyping of zebrafish. We present a new automated method that is able to detect blob objects, such as nuclei or cells in microscopic zebrafish images. This method is composed of three key steps. The first step is to produce a diffused gradient vector field by a physical elastic deformable model. In the second step, the flux image is computed on the diffused gradient vector field. The third step performs thresholding and nonmaximum suppression based on the flux image. We report the validation and experimental results of this method using zebrafish image datasets from three independent research labs. Both sensitivity and specificity of this method are over 90%. This method is able to differentiate closely juxtaposed or connected blob objects, with high sensitivity and specificity in different situations. It is characterized by a good, consistent performance in blob object detection.

Development and automation of a test of impulse control in zebrafish

PubMed Central

Parker, Matthew O.; Ife, Dennis; Ma, Jun; Pancholi, Mahesh; Smeraldi, Fabrizio; Straw, Chris; Brennan, Caroline H.

2013-01-01

Deficits in impulse control (difficulties in inhibition of a pre-potent response) are fundamental to a number of psychiatric disorders, but the molecular and cellular basis is poorly understood. Zebrafish offer a very useful model for exploring these mechanisms, but there is currently a lack of validated procedures for measuring impulsivity in fish. In mammals, impulsivity can be measured by examining rates of anticipatory responding in the 5-choice serial reaction time task (5-CSRTT), a continuous performance task where the subject is reinforced upon accurate detection of a briefly presented light in one of five distinct spatial locations. This paper describes the development of a fully-integrated automated system for testing impulsivity in adult zebrafish. We outline the development of our image analysis software and its integration with National Instruments drivers and actuators to produce the system. We also describe an initial validation of the system through a one-generation screen of chemically mutagenized zebrafish, where the testing parameters were optimized. PMID:24133417

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

PubMed Central

Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

2017-01-01

Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

PubMed Central

Kim, Seok-Hyung; Kowalski, Marie L.; Carson, Robert P.; Bridges, L. Richard; Ess, Kevin C.

2013-01-01

SUMMARY Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors. PMID:23580196

Triclosan is a Mitochondrial Uncoupler in Live Zebrafish

PubMed Central

Shim, Juyoung; Weatherly, Lisa M.; Luc, Richard H.; Dorman, Maxwell T.; Neilson, Andy; Ng, Ryan; Kim, Carol H.; Millard, Paul J.; Gosse, Julie A.

2016-01-01

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24 hour post fertilization zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, following acute exposure to TCS, basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96 well assay format. The method developed in this study provides a high-throughput tool to identify previously-unknown mitochondrial uncouplers in a living organism. PMID:27111768

High resolution fate map of the zebrafish diencephalon.

PubMed

Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

2009-07-01

The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described. (c) 2009 Wiley-Liss, Inc.

Quantification of locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies.

PubMed

Ingebretson, Justin J; Masino, Mark A

2013-01-01

High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

PubMed

Poon, Kar Lai; Wang, Xingang; Lee, Serene G P; Ng, Ashley S; Goh, Wei Huang; Zhao, Zhonghua; Al-Haddawi, Muthafar; Wang, Haishan; Mathavan, Sinnakaruppan; Ingham, Philip W; McGinnis, Claudia; Carney, Tom J

2017-03-01

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Dietary Strontium Increases Bone Mineral Density in Intact Zebrafish (Danio rerio): A Potential Model System for Bone Research

PubMed Central

Padgett-Vasquez, Steve; Garris, Heath W.; Nagy, Tim R.; D'Abramo, Louis R.; Watts, Stephen A.

2010-01-01

Abstract Zebrafish (Danio rerio) skeletal bone possesses properties similar to human bone, which suggests that they may be used as a model to study mineralization characteristics of the human Haversian system, as well as human bone diseases. One prerequisite for the use of zebrafish as an alternative osteoporotic bone model is to determine whether their bone displays functional plasticity similar to that observed in other bone models. Strontium citrate was supplemented into a laboratory-prepared diet (45% crude protein) to produce dietary strontium levels of 0%, 0.63%, 1.26%, 1.89%, and 2.43% and fed ad libitum twice daily for 12 weeks to 28-day-old intact zebrafish. Length was determined at 4-week intervals, and both weight and length were recorded at 12 weeks. At 12 weeks, seven zebrafish from each dietary level were analyzed for total bone mineral density by microcomputed tomography. Dietary strontium citrate supplementation significantly (p < 0.05) increased zebrafish whole-body and spinal column bone mineral density. In addition, trace amounts of strontium were incorporated into the scale matrix in those zebrafish that consumed strontium-supplemented diets. These findings suggest that zebrafish bone displays plasticity similar to that reported for other bone models (i.e., rat, mouse, and monkey) that received supplements of strontium compounds and zebrafish should be viewed as an increasingly valuable bone model. PMID:20874492

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

PubMed

Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

2016-01-01

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Automated Planar Tracking the Waving Bodies of Multiple Zebrafish Swimming in Shallow Water

PubMed Central

Wang, Shuo Hong; Cheng, Xi En; Qian, Zhi-Ming; Liu, Ye; Chen, Yan Qiu

2016-01-01

Zebrafish (Danio rerio) is one of the most widely used model organisms in collective behavior research. Multi-object tracking with high speed camera is currently the most feasible way to accurately measure their motion states for quantitative study of their collective behavior. However, due to difficulties such as their similar appearance, complex body deformation and frequent occlusions, it is a big challenge for an automated system to be able to reliably track the body geometry of each individual fish. To accomplish this task, we propose a novel fish body model that uses a chain of rectangles to represent fish body. Then in detection stage, the point of maximum curvature along fish boundary is detected and set as fish nose point. Afterwards, in tracking stage, we firstly apply Kalman filter to track fish head, then use rectangle chain fitting to fit fish body, which at the same time further judge the head tracking results and remove the incorrect ones. At last, a tracklets relinking stage further solves trajectory fragmentation due to occlusion. Experiment results show that the proposed tracking system can track a group of zebrafish with their body geometry accurately even when occlusion occurs from time to time. PMID:27128096

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish.

PubMed

Gamse, Joshua T; Gorelick, Daniel A

2016-10-01

For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.

Developmental Toxicity of the Organic Fraction from Hydraulic Fracturing Flowback and Produced Waters to Early Life Stages of Zebrafish ( Danio rerio).

PubMed

He, Yuhe; Sun, Chenxing; Zhang, Yifeng; Folkerts, Erik J; Martin, Jonathan W; Goss, Greg G

2018-03-20

Hydraulic fracturing (HF) has emerged as a major recovery method of unconventional oil and gas reservoirs and concerns have been raised regarding the environmental impact of releases of Flowback and Produced Water (FPW) to aquatic ecosystems. To investigate potential effects of HF-FPW on fish embryo development, HF-FPW samples were collected from two different wells and the organic fractions were isolated from both aqueous and particle phases to eliminate the confounding effects of high salinity. Each organic extract was characterized by non-target analysis with HPLC-Orbitrap-MS, with targeted analysis for polycyclic aromatic hydrocarbons provided as markers of petroleum-affected water. The organic profiles differed between samples, including PAHs and alkyl PAHs, and major substances identified by non-target analysis included polyethylene glycols, alkyl ethoxylates, octylphenol ethoxylates, and other high molecular weight (C 49-79 ) ethylene oxide polymeric material. Zebrafish embryos were exposed to various concentrations of FPW organic extracts to investigate acute (7-day) and developmental toxicity in early life stages. The acute toxicity (LD 50 ) of the extracted FPW fractions ranged from 2.8× to 26× the original organic content. Each extracted FPW fraction significantly increased spinal malformation, pericardial edema, and delayed hatch in exposed embryos and altered the expression of a suite of target genes related to biotransformation, oxidative stress, and endocrine-mediation in developing zebrafish embryos. These results provide novel information on the variation of organic profiles and developmental toxicity among different sources and fractions of HF-FPWs.

Mycobacteriosis in zebrafish colonies.

PubMed

Whipps, Christopher M; Lieggi, Christine; Wagner, Robert

2012-01-01

Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

Episodic-like memory in zebrafish.

PubMed

Hamilton, Trevor J; Myggland, Allison; Duperreault, Erika; May, Zacnicte; Gallup, Joshua; Powell, Russell A; Schalomon, Melike; Digweed, Shannon M

2016-11-01

Episodic-like memory tests often aid in determining an animal's ability to recall the what, where, and which (context) of an event. To date, this type of memory has been demonstrated in humans, wild chacma baboons, corvids (Scrub jays), humming birds, mice, rats, Yucatan minipigs, and cuttlefish. The potential for this type of memory in zebrafish remains unexplored even though they are quickly becoming an essential model organism for the study of a variety of human cognitive and mental disorders. Here we explore the episodic-like capabilities of zebrafish (Danio rerio) in a previously established mammalian memory paradigm. We demonstrate that when zebrafish were presented with a familiar object in a familiar context but a novel location within that context, they spend more time in the novel quadrant. Thus, zebrafish display episodic-like memory as they remember what object they saw, where they saw it (quadrant location), and on which occasion (yellow or blue walls) it was presented.

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish

PubMed Central

Gamse, Joshua T.

2016-01-01

Abstract For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals. PMID:27618129

Escherichia coli O78 isolated from septicemic lambs shows high pathogenicity in a zebrafish model.

PubMed

Kjelstrup, Cecilie K; Barber, Amelia E; Norton, J Paul; Mulvey, Matthew A; L'Abée-Lund, Trine M

2017-01-25

The pathogenicity of Escherichia coli O78 strain K46, originally isolated from an outbreak of septicemia in neonatal lambs, was investigated in zebrafish embryo and murine models of infection. Its biofilm potential, cellulose production, and the expression of type 1 pili and curli fimbriae were measured by in vitro assays. The strain was highly pathogenic in the zebrafish embryo model of infection, where it killed all embryos within 24 h post inoculation (hpi) at doses as low as 1000 colony forming units. Zebrafish embryos inoculated with similar doses of commensal E. coli strains showed no signs of disease, and cleared the bacteria within 24 h. E. coli K46 colonized the murine gut at the same level as the uropathogenic E. coli (UPEC) reference strain CFT073 in CBA/J mice after oral inoculation, but infected the murine bladder significantly less than CFT073 after transurethral inoculation. Type 1 pili were clearly expressed by E. coli K46, while curli fimbriae and cellulose production were weakly expressed. The ability to produce biofilm varied in different growth media, but overall E. coli K46 was a poorer biofilm producer compared to the reference strain E. coli UTI89. In conclusion, the zebrafish lethality model provides further evidence that E. coli K46 is highly pathogenic and might be useful in future studies to identify bacterial virulence factors.

Zebrafish model systems for developmental neurobehavioral toxicology.

PubMed

Bailey, Jordan; Oliveri, Anthony; Levin, Edward D

2013-03-01

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. Copyright © 2013 Wiley Periodicals, Inc.

Zebrafish Model Systems for Developmental Neurobehavioral Toxicology

PubMed Central

Bailey, Jordan; Oliveri, Anthony; Levin, Edward D.

2014-01-01

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. PMID:23723169

Live imaging reveals a conserved role of fatty acid β-oxidation in early lymphatic development in zebrafish.

PubMed

Zecchin, Annalisa; Wong, Brian W; Tembuyser, Bieke; Souffreau, Joris; Van Nuffelen, An; Wyns, Sabine; Vinckier, Stefan; Carmeliet, Peter; Dewerchin, Mieke

2018-06-18

During embryonic development, lymphatic endothelial cells (LECs) differentiate from venous endothelial cells (VECs), a process that is tightly regulated by several genetic signals. While the aquatic zebrafish model is regularly used for studying lymphangiogenesis and offers the unique advantage of time-lapse video-imaging of lymphatic development, some aspects of lymphatic development in this model differ from those in the mouse. It therefore remained to be determined whether fatty acid β-oxidation (FAO), which we showed to regulate lymphatic formation in the mouse, also co-determines lymphatic development in this aquatic model. Here, we took advantage of the power of the zebrafish embryo model to visualize the earliest steps of lymphatic development through time-lapse video-imaging. By targeting zebrafish isoforms of carnitine palmitoyltransferase 1a (cpt1a), a rate controlling enzyme of FAO, with multiple morpholinos, we demonstrate that reducing CPT1A levels and FAO flux during zebrafish development impairs lymphangiogenic secondary sprouting, the initiation of lymphatic development in the zebrafish trunk, and the formation of the first lymphatic structures. These findings not only show evolutionary conservation of the importance of FAO for lymphatic development, but also suggest a role for FAO in co-regulating the process of VEC-to-LEC differentiation in zebrafish in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

A zebrafish model of diabetes mellitus and metabolic memory.

PubMed

Intine, Robert V; Olsen, Ansgar S; Sarras, Michael P

2013-02-28

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and

From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

PubMed Central

Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

2012-01-01

The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

Learning and memory in zebrafish (Danio rerio).

PubMed

Gerlai, R

2016-01-01

Learning and memory are defining features of our own species inherently important to our daily lives and to who we are. Without our memories we cease to exist as a person. Without our ability to learn individuals and collectively our society would cease to function. Diseases of the mind still remain incurable. The interest in understanding of the mechanisms of learning and memory is thus well founded. Given the complexity of such mechanisms, concerted efforts have been made to study them under controlled laboratory conditions, ie, with laboratory model organisms. The zebrafish, although new in this field, is one such model organism. The rapidly developing forward- and reverse genetic methods designed for the zebrafish and the increasing use of pharmacological tools along with numerous neurobiology techniques make this species perhaps the best model for the analysis of the mechanisms of complex central nervous system characteristics. The fact that it is an evolutionarily ancient and simpler vertebrate, but at the same time it possesses numerous conserved features across multiple levels of biological organization makes this species an excellent tool for the analysis of the mechanisms of learning and memory. The bottleneck lies in our understanding of its cognitive and mnemonic features, the topic of this chapter. The current paper builds on a chapter published in the previous edition and continues to focus on associative learning, but now it extends the discussion to other forms of learning and to recent discoveries on memory-related features and findings obtained both in adults and larval zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of ZnSO4-induced peripheral anosmia on zebrafish behavior and physiology.

PubMed

Abreu, Murilo S; Giacomini, Ana C V V; Rodriguez, Rubens; Kalueff, Allan V; Barcellos, Leonardo J G

2017-03-01

Olfaction plays a key role in modulating behavioral and physiological responses of various animal species, including fishes. Olfactory deficits can be induced in fish experimentally, and utilized to examine the role of olfaction in their normal and pathological behaviors. Here, we examine whether experimental anosmia, evoked by ZnSO 4 in adult zebrafish can be associated with behavioral and/or physiological responses. We show that experimental ZnSO 4 -induced anosmia caused acute, but not prolonged, anxiogenic-like effects on zebrafish behavior tested in the novel tank test. The procedure also elevated whole-body cortisol levels in zebrafish. Moreover, ZnSO4 treatment, but not sham, produced damage to olfactory epithelium, inducing overt basal cell vacuolization and intercellular edema. The loss of olfaction, assessed by the fish food preference behavior in the aquatic Y-maze, was present 1h, but not 24h, after the treatment. Collectively, this suggests that transient experimental anosmia by ZnSO 4 modulates zebrafish behavior and olfaction, which can be used to evoke and assess their stress-related anxiety-like states. Copyright © 2016 Elsevier B.V. All rights reserved.

Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

PubMed Central

Yoshida, Nagisa; Frickel, Eva-Maria; Mostowy, Serge

2017-01-01

Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans. PMID:29250076

Use of Zebrafish Larvae as a Multi-Endpoint Platform to Characterize the Toxicity Profile of Silica Nanoparticles.

PubMed

Pham, Duc-Hung; De Roo, Bert; Nguyen, Xuan-Bac; Vervaele, Mattias; Kecskés, Angela; Ny, Annelii; Copmans, Daniëlle; Vriens, Hanne; Locquet, Jean-Pierre; Hoet, Peter; de Witte, Peter A M

2016-11-22

Nanomaterials are being extensively produced and applied in society. Human and environmental exposures are, therefore, inevitable and so increased attention is being given to nanotoxicity. While silica nanoparticles (NP) are one of the top five nanomaterials found in consumer and biomedical products, their toxicity profile is poorly characterized. In this study, we investigated the toxicity of silica nanoparticles with diameters 20, 50 and 80 nm using an in vivo zebrafish platform that analyzes multiple endpoints related to developmental, cardio-, hepato-, and neurotoxicity. Results show that except for an acceleration in hatching time and alterations in the behavior of zebrafish embryos/larvae, silica NPs did not elicit any developmental defects, nor any cardio- and hepatotoxicity. The behavioral alterations were consistent for both embryonic photomotor and larval locomotor response and were dependent on the concentration and the size of silica NPs. As embryos and larvae exhibited a normal touch response and early hatching did not affect larval locomotor response, the behavior changes observed are most likely the consequence of modified neuroactivity. Overall, our results suggest that silica NPs do not cause any developmental, cardio- or hepatotoxicity, but they pose a potential risk for the neurobehavioral system.

Inheritance patterns of morphological laterality in mouth opening of zebrafish, Danio rerio.

PubMed

Hata, Hiroki; Hori, Michio

2012-01-01

The inheritance patterns of asymmetry in mouth opening in zebrafish were investigated using crossing experiments. Zebrafish exhibit asymmetric laterality in mouth opening, with each individual having either a leftward (righty) or rightward (lefty) bias. All righty incrosses produced only righty F(1), whereas all lefty incrosses resulted in an F(1) L:R ratio of 2:1. All test crosses between lefty and righty individuals resulted in an F(1) L:R=1:1. These results were consistent with the hereditary pattern for Japanese medaka, three Tanganyikan cichlids, and a Japanese riverine goby. The pattern suggests a one-locus two-allele Mendelian model of inheritance, with the lefty allele being dominant over righty and the dominant homozygote being lethal. To determine the reason for the absence of lefty homozygotes, the survival rates of the offspring were examined according to developmental stage. Survival did not differ among combinations of parent laterality. Thus the mechanism underlying the lethality of the dominant homozygote remains unclear. This study showed that the mouth-opening laterality of zebrafish is genetically determined and that the direction follows a Mendelian inheritance pattern that is shared among cypriniform zebrafish, beloniform medaka, perciform cichlids, and a goby, suggesting a common genetic background in mouth-opening laterality among these species.

Contextual Fear Conditioning in Zebrafish

ERIC Educational Resources Information Center

Kenney, Justin W.; Scott, Ian C.; Josselyn, Sheena A.; Frankland, Paul W.

2017-01-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development…

Triclosan is a mitochondrial uncoupler in live zebrafish.

PubMed

Shim, Juyoung; Weatherly, Lisa M; Luc, Richard H; Dorman, Maxwell T; Neilson, Andy; Ng, Ryan; Kim, Carol H; Millard, Paul J; Gosse, Julie A

2016-12-01

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here, we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24-hour post-fertilization (hpf) zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XF e 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, after acute exposure to TCS, the basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96-well assay format. The method developed in this study provides a high-throughput tool to identify previously unknown mitochondrial uncouplers in a living organism. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Vitamin D receptor deficiency impairs inner ear development in zebrafish.

PubMed

Kwon, Hye-Joo

2016-09-16

The biological actions of vitamin D are largely mediated through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, which regulates gene expression in a wide variety of tissues and cells. Mutations in VDR gene have been implicated in ear disorders (hearing loss and balance disorder) but the mechanisms are not well established. In this study, to investigate the role of VDR in inner ear development, morpholino-mediated gene knockdown approaches were used in zebrafish model system. Two paralogs for VDR, vdra and vdrb, have been identified in zebrafish. Knockdown of vdra had no effect on ear development, whereas knockdown of vdrb displayed morphological ear defects including smaller otic vesicles with malformed semicircular canals and abnormal otoliths. Loss-of-vdrb resulted in down-regulation of pre-otic markers, pax8 and pax2a, indicating impairment of otic induction. Furthermore, zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination. These data reveal that VDR signaling plays an important role in ear development. Copyright © 2016 Elsevier Inc. All rights reserved.

Knockdown of prothrombin in zebrafish.

PubMed

Day, Kenneth; Krishnegowda, Naveen; Jagadeeswaran, Pudur

2004-01-01

Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.

Juxtaposition of chemical and mutation-induced developmental defects in zebrafish reveal a copper-chelating activity for kalihinol F.

PubMed

Sandoval, Imelda T; Manos, Elizabeth J; Van Wagoner, Ryan M; Delacruz, Richard Glenn C; Edes, Kornelia; Winge, Dennis R; Ireland, Chris M; Jones, David A

2013-06-20

A major hurdle in using complex systems for drug screening is the difficulty of defining the mechanistic targets of small molecules. The zebrafish provides an excellent model system for juxtaposing developmental phenotypes with mechanism discovery using organism genetics. We carried out a phenotype-based screen of uncharacterized small molecules in zebrafish that produced a variety of chemically induced phenotypes with potential genetic parallels. Specifically, kalihinol F caused an undulated notochord, defects in pigment formation, hematopoiesis, and neural development. These phenotypes were strikingly similar to the zebrafish mutant, calamity, an established model of copper deficiency. Further studies into the mechanism of action of kalihinol F revealed a copper-chelating activity. Our data support this mechanism of action for kalihinol F and the utility of zebrafish as an effective system for identifying therapeutic and target pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

PubMed

Zhu, Xiao-Yu; Wu, Si-Qi; Guo, Sheng-Ya; Yang, Hua; Xia, Bo; Li, Ping; Li, Chun-Qi

2018-03-20

Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 μM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

New isocoumarins from a cold-adapted fungal strain mucor sp. and their developmental toxicity to zebrafish embryos.

PubMed

Feng, Chun-Chi; Chen, Guo-Dong; Zhao, Yan-Qiu; Xin, Sheng-Chang; Li, Song; Tang, Jin-Shan; Li, Xiao-Xia; Hu, Dan; Liu, Xing-Zhong; Gao, Hao

2014-07-01

Three new isocoumarin derivatives, mucorisocoumarins A-C (1-3, resp.), together with seven known compounds, 4-10, were isolated from the cold-adapted fungal strain Mucor sp. (No. XJ07027-5). The structures of the new compounds were identified by detailed IR, MS, and 1D- and 2D-NMR analyses. It was noteworthy that compounds 1, 2, 4, and 5 were successfully resolved by chiral HPLC, indicating that 1-7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Zebrafish: an animal model for research in veterinary medicine.

PubMed

Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

2015-01-01

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic

Complex cardiac defects after ethanol exposure during discrete cardiogenic events in zebrafish: Prevention with folic acid

PubMed Central

Sarmah, Swapnalee; Marrs, James A.

2014-01-01

BACKGROUND Fetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. RESULTS Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbed multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. CONCLUSIONS Our results indicate that ethanol exposure interrupted divergent cardiac morphogenesis events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects. PMID:23832875

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

PubMed Central

Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

2015-01-01

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.

PubMed

Chou, Chih-Ming; Chen, Yi-Chung; Lee, Ming-Ting; Chen, Gen-Der; Lu, I-Ching; Chen, Shui-Tsung; Huang, Chang-Jen

2006-02-17

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.

Zebrafish: A Versatile Animal Model for Fertility Research.

PubMed

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

Zebrafish: A Versatile Animal Model for Fertility Research

PubMed Central

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

2016-01-01

The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research. PMID:27556045

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN.

PubMed

Noyes, Pamela D; Garcia, Gloria R; Tanguay, Robert L

2016-12-21

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world

ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN

PubMed Central

Noyes, Pamela D.; Garcia, Gloria R.; Tanguay, Robert L.

2016-01-01

Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish

PubMed Central

Rovira, Mireia; Arrey, Gerard; Planas, Josep V.

2017-01-01

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a β-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic

Physical exercise improves learning in zebrafish, Danio rerio.

PubMed

Luchiari, Ana Carolina; Chacon, Diana Marques Martins

2013-11-01

Zebrafish is an ideal vertebrate model for neuroscience studies focusing on learning and memory. Although genetic manipulation of zebrafish is available, behavioral protocols are often lacking. In this study we tested whether physical activity can facilitate zebrafish's learning process in an associative conditioning task. Learning was inferred by the approach of the feeding area just after the conditioned stimulus (light). Unexercised zebrafish showed conditioning response from the 5th testing day while fish previously submitted to swim against the water current showed learning by the 3rd day of testing. It seems that physical activity may accelerate associative learning response in zebrafish, indicating the benefits of exercise for cognitive processes. We suggest that this preliminary work could be useful for high throughput screening. Copyright © 2013 Elsevier B.V. All rights reserved.

Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

PubMed

Cachat, Jonathan; Kyzar, Evan J; Collins, Christopher; Gaikwad, Siddharth; Green, Jeremy; Roth, Andrew; El-Ounsi, Mohamed; Davis, Ari; Pham, Mimi; Landsman, Samuel; Stewart, Adam Michael; Kalueff, Allan V

2013-01-01

An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

PubMed Central

2011-01-01

Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome. PMID:22111588

Normal anatomy and histology of the adult zebrafish.

PubMed

Menke, Aswin L; Spitsbergen, Jan M; Wolterbeek, Andre P M; Woutersen, Ruud A

2011-08-01

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

PubMed

Spagnoli, Sean; Xue, Lan; Kent, Michael L

2015-09-15

The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the

Making Waves: New Developments in Toxicology With the Zebrafish.

PubMed

Horzmann, Katharine A; Freeman, Jennifer L

2018-05-01

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

PubMed

Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

2017-10-01

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

Learning and memory in zebrafish larvae

PubMed Central

Roberts, Adam C.; Bill, Brent R.; Glanzman, David L.

2013-01-01

Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory. PMID:23935566

Shp2–Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration

PubMed Central

Hale, Alexander James

2017-01-01

ABSTRACT Regeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a−/− ptpn11b−/− zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a−/− ptpn11b−/− zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a–mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration. PMID:29203641

Quantification of locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies

PubMed Central

Ingebretson, Justin J.; Masino, Mark A.

2013-01-01

High-throughput behavioral studies using larval zebrafish often assess locomotor activity to determine the effects of experimental perturbations. However, the results reported by different groups are difficult to compare because there is not a standardized experimental paradigm or measure of locomotor activity. To address this, we investigated the effects that several factors, including the stage of larval development and the physical dimensions (depth and diameter) of the behavioral arena, have on the locomotor activity produced by larval zebrafish. We provide evidence for differences in locomotor activity between larvae at different stages and when recorded in wells of different depths, but not in wells of different diameters. We also show that the variability for most properties of locomotor activity is less for older than younger larvae, which is consistent with previous reports. Finally, we show that conflicting interpretations of activity level can occur when activity is assessed with a single measure of locomotor activity. Thus, we conclude that although a combination of factors should be considered when designing behavioral experiments, the use of older larvae in deep wells will reduce the variability of locomotor activity, and that multiple properties of locomotor activity should be measured to determine activity level. PMID:23772207

Development of sensory systems in zebrafish (Danio rerio)

NASA Technical Reports Server (NTRS)

Moorman, S. J.

2001-01-01

Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

PubMed Central

Hui, Subhra Prakash; Nag, Tapas Chandra; Ghosh, Sukla

2015-01-01

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration. PMID:26630262

UNUSUAL FINDINGS IN ZEBRAFISH, DANIO RERIO, FROM TOXICOLOGICAL STUDIES AND THE ZEBRAFISH INTERNATIONAL RESOURCE CENTER DIAGNOSTIC SERVICE

EPA Science Inventory

A number of interesting and unusual lesions have been diagnosed in zebrafish that have been evaluated from toxicological studies or submitted as cases to the Diagnostic Service at Oregon State University. Lesions were observed in various wild-type and mutant lines of zebrafish an...

Contextual fear conditioning in zebrafish.

PubMed

Kenney, Justin W; Scott, Ian C; Josselyn, Sheena A; Frankland, Paul W

2017-10-01

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory. © 2017 Kenney et al.; Published by Cold Spring Harbor Laboratory Press.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

PubMed

Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

2006-06-15

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

PubMed

Nema, Shubham; Bhargava, Yogesh

2016-08-01

Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.

Home tank water versus novel water differentially affect alcohol-induced locomotor activity and anxiety related behaviours in zebrafish.

PubMed

Tran, Steven; Facciol, Amanda; Gerlai, Robert

2016-05-01

The zebrafish may be uniquely well suited for studying alcohol's mechanisms of action in vivo, since alcohol can be administered via immersion in a non-invasive manner. Despite the robust behavioural effects of alcohol administration in mammals, studies reporting the locomotor stimulant and anxiolytic effects of alcohol in zebrafish have been inconsistent. In the current study, we examined whether differences in the type of water used for alcohol exposure and behavioural testing contribute to these inconsistencies. To answer this question, we exposed zebrafish to either home water from their housing tanks or novel water from an isolated reservoir (i.e. water lacking zebrafish chemosensory and olfactory cues) with 0% or 1% v/v alcohol for 30 min, a 2 × 2 between subject experimental designs. Behavioural responses were quantified throughout the 30-minute exposure session via a video tracking system. Although control zebrafish exposed to home water and novel water were virtually indistinguishable in their behavioural responses, alcohol's effect on locomotor activity and anxiety-like behavioural responses were dependent on the type of water used for testing. Alcohol exposure in home tank water produced a mild anxiolytic and locomotor stimulant effect, whereas alcohol exposure in novel water produced an anxiogenic effect without altering locomotor activity. These results represent a dissociation between alcohol's effects on locomotor and anxiety related responses, and also illustrate how environmental factors, in this case familiarity with the water, may interact with such effects. In light of these findings, we urge researchers to explicitly state the type of water used. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental stage.

PubMed

Zhu, Xiaoshan; Zhu, Lin; Duan, Zhenghua; Qi, Ruiqi; Li, Yan; Lang, Yupeng

2008-02-15

With the emergence of manufactured nanomaterials, it is urgent to carry out researches on their potential environmental impacts and biological effects. To better understand the potential ecotoxicological impacts of metal oxide nanoparticles released to aquatic environments, the zebrafish 96-h embryo-larval bioassay was used to assess and compare the developmental toxicities of nanoscale zinc oxide (nZnO), titanium dioxide (nTiO(2)) and alumina (nAl(2)O(3)) aqueous suspensions. Toxicological endpoints such as zebrafish embryos or larvae survival, hatching rate and malformation were noted and described within 96 h of exposure. Meanwhile, a comparative experiment with their bulk counterparts (i.e., ZnO/bulk, TiO(2)/bulk and Al(2)O(3)/bulk) was conducted to understand the effect of particle size on their toxicities. The results showed that: (i) both nZnO and ZnO/bulk aqueous suspensions delayed zebrafish embryo and larva development, decreased their survival and hatching rates, and caused tissue damage. The 96-h LC(50) of nZnO and ZnO/bulk aqueous suspensions on the zebrafish survival are 1.793 mg/L and 1.550 mg/L respectively; and the 84-h EC(50) on the zebrafish embryo hatching rate are 2.065 mg/L and 2.066 mg/L respectively. Serious tissue ulceration was found on zebrafish larvae exposed to nZnO and ZnO/bulk aqueous suspensions. (ii) In contrast, neither nTiO(2) and TiO(2)/bulk nor nAl(2)O(3) and Al(2)O(3)/bulk showed any toxicity to zebrafish embryos and larvae under the same experimental condition. It revealed that the metal oxide nanoparticles with different chemical composition have different zebrafish developmental toxicities. (iii) Exposures of nTiO(2), nZnO and nAl(2)O(3) produced toxic effects on zebrafish embryos and larvae, which was not different from the effects caused by exposing to their bulk counterparts. This is the first study about the developmental toxicity of metal oxide nanoparticles, and the results demonstrate that nZnO is very toxic to

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

PubMed Central

Wong, Ten-Tsao; Collodi, Paul

2013-01-01

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock. PMID:23826390

Genomic Approaches to Zebrafish Cancer

PubMed Central

2017-01-01

The zebrafish has emerged as an important model for studying cancer biology. Identification of DNA, RNA and chromatin abnormalities can give profound insight into the mechanisms of tumorigenesis and the there are many techniques for analyzing the genomes of these tumors. Here, I present an overview of the available technologies for analyzing tumor genomes in the zebrafish, including array based methods as well as next-generation sequencing technologies. I also discuss the ways in which zebrafish tumor genomes can be compared to human genomes using cross-species oncogenomics, which act to filter genomic noise and ultimately uncover central drivers of malignancy. Finally, I discuss downstream analytic tools, including network analysis, that can help to organize the alterations into coherent biological frameworks that can then be investigated further. PMID:27165352

Transgene manipulation in zebrafish by using recombinases.

PubMed

Dong, Jie; Stuart, Gary W

2004-01-01

Although much remains to be done, our results to date suggest that efficient and precise genome engineering in zebrafish will be possible in the future by using Cre recombinase and SB transposase in combination with their respective target sites. In this study, we provide the first evidence that Cre recombinase can mediate effective site-specific deletion of transgenes in zebrafish. We found that the efficiency of target site utilization could approach 100%, independent of whether the target site was provided transiently by injection or stably within an integrated transgene. Microinjection of Cre mRNA appeared to be slightly more effective for this purpose than microinjection of Cre-expressing plasmid DNA. Our work has not yet progressed to the point where SB-mediated mobilization of our transgene constructs would be observed. However, a recent report has demonstrated that SB can enhance transgenesis rates sixfold over conventional methods by efficiently mediating multiple single-copy insertion of transgenes into the zebrafish genome (Davidson et al., 2003). Therefore, it seems likely that a combined system should eventually allow both SB-mediated transgene mobilization and Cre-mediated transgene modification. Our goal is to validate methods for the precise reengineering of the zebrafish genome by using a combination of Cre-loxP and SB transposon systems. These methods can be used to delete, replace, or mobilize large pieces of DNA or to modify the genome only when and where required by the investigator. For example, it should be possible to deliver particular RNAi genes to well-expressed chromosomal loci and then exchange them easily with alternative RNAi genes for the specific suppression of alternative targets. As a nonviral vector for gene therapy, the transposon component allows for the possibility of highly efficient integration, whereas the Cre-loxP component can target the integration and/or exchange of foreign DNA into specific sites within the genome. The

In vivo cell tracking and quantification method in adult zebrafish

NASA Astrophysics Data System (ADS)

Zhang, Li; Alt, Clemens; Li, Pulin; White, Richard M.; Zon, Leonard I.; Wei, Xunbin; Lin, Charles P.

2012-03-01

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Zebrafish tracking using convolutional neural networks.

PubMed

Xu, Zhiping; Cheng, Xi En

2017-02-17

Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

Zebrafish tracking using convolutional neural networks

NASA Astrophysics Data System (ADS)

Xu, Zhiping; Cheng, Xi En

2017-02-01

Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

Zebrafish as model organisms for studying drug-induced liver injury

PubMed Central

Vliegenthart, A D Bastiaan; Tucker, Carl S; Del Pozo, Jorge; Dear, James W

2014-01-01

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers. PMID:24773296

Identification and expression analysis of zebrafish glypicans during embryonic development.

PubMed

Gupta, Mansi; Brand, Michael

2013-01-01

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

Heart Repair and Regeneration: Recent Insights from Zebrafish Studies

PubMed Central

Lien, Ching-Ling; Harrison, Michael R.; Tuan, Tai-Lan; Starnes, Vaughn A

2012-01-01

Cardiovascular disease is the leading cause of death in United States and worldwide. Failure to properly repair or regenerate damaged cardiac tissues after myocardial infarction is a major cause of heart failure. In contrast to humans and other mammals, zebrafish hearts regenerate after substantial injury or tissue damage. Here, we review recent progress in studying zebrafish heart regeneration, addressing the molecular and cellular responses in the three tissue layers of the heart: myocardium, epicardium, and endocardium. We also compare different injury models utilized to study zebrafish heart regeneration, and discuss the differences in responses to injury between mammalian and zebrafish hearts. By learning how zebrafish hearts regenerate naturally, we can better design therapeutic strategies for repairing human hearts after myocardial infarction. PMID:22818295

The zebrafish genome: a review and msx gene case study.

PubMed

Postlethwait, J H

2006-01-01

Zebrafish is one of several important teleost models for understanding principles of vertebrate developmental, molecular, organismal, genetic, evolutionary, and genomic biology. Efficient investigation of the molecular genetic basis of induced mutations depends on knowledge of the zebrafish genome. Principles of zebrafish genomic analysis, including gene mapping, ortholog identification, conservation of syntenies, genome duplication, and evolution of duplicate gene function are discussed here using as a case study the zebrafish msxa, msxb, msxc, msxd, and msxe genes, which together constitute zebrafish orthologs of tetrapod Msx1, Msx2, and Msx3. Genomic analysis suggests orthologs for this difficult to understand group of paralogs.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center

PubMed Central

Varga, Zoltán M.; Kent, Michael L.

2016-01-01

Abstract The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers. PMID:27031282

Advancing epilepsy treatment through personalized genetic zebrafish models.

PubMed

Griffin, A; Krasniak, C; Baraban, S C

2016-01-01

With an increase in the number of disease causing genetic mutations identified from epilepsy cohorts, zebrafish are proving to be an attractive vertebrate model for functional analysis of these allele variants. Not only do zebrafish have conserved gene functions, but larvae harboring mutations in identified human epileptic genes show spontaneous seizure activity and mimic the convulsive behavioral movements observed in humans. With zebrafish being compatible with medium to high-throughput screening, they are also proving to be a unique and powerful system for early preclinical drug screening, including novel target identification, pharmacology, and toxicology. Additionally, with recent advances in genomic engineering technologies, it is now possible to study the precise pathophysiology of patient-specific gene mutations in zebrafish. The following sections highlight how the unique attributes of zebrafish, in combination with genetic modifications, are continuing to transform our understanding of epilepsy and help identify personalized therapeutics for specific patient cohorts. © 2016 Elsevier B.V. All rights reserved.

Examination of a Palatogenic Gene Program in Zebrafish

PubMed Central

Swartz, Mary E.; Sheehan-Rooney, Kelly; Dixon, Michael J.; Eberhart, Johann K.

2011-01-01

Human palatal clefting is debilitating and difficult to rectify surgically. Animal models enhance our understanding of palatogenesis and are essential in strategies designed to ameliorate palatal malformations in humans. Recent studies have shown that the zebrafish palate, or anterior neurocranium, is under similar genetic control to the amniote palatal skeleton. We extensively analyzed palatogenesis in zebrafish to determine the similarity of gene expression and function across vertebrates. By 36 hpf palatogenic cranial neural crest cells reside in homologous regions of the developing face compared to amniote species. Transcription factors and signaling molecules regulating mouse palatogenesis are expressed in similar domains during palatogenesis in zebrafish. Functional investigation of a subset of these genes, fgf10a, tgfb2, pax9 and smad5 revealed their necessity in zebrafish palatogenesis. Collectively, these results suggest that the gene regulatory networks regulating palatogenesis may be conserved across vertebrate species, demonstrating the utility of zebrafish as a model for palatogenesis. PMID:22016187

Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

PubMed

Panier, Thomas; Romano, Sebastián A; Olive, Raphaël; Pietri, Thomas; Sumbre, Germán; Candelier, Raphaël; Debrégeas, Georges

2013-01-01

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

Multiple myeloma presenting as CEA-producing rectal cancer.

PubMed

Talamo, Giampaolo; Barochia, Amitkumar; Zangari, Maurizio; Loughran, Thomas P

2010-03-31

We report the case of a 57-year-old patient with multiple myeloma, characterized by extramedullary involvement of the rectum at presentation. Malignant plasma cells were found to produce carcinoembryonic antigen (CEA), a tumor antigen more commonly associated with rectal adenocarcinomas.

Systematic approaches to toxicology in the zebrafish.

PubMed

Peterson, Randall T; Macrae, Calum A

2012-01-01

As the current paradigms of drug discovery evolve, it has become clear that a more comprehensive understanding of the interactions between small molecules and organismal biology will be vital. The zebrafish is emerging as a complement to existing in vitro technologies and established preclinical in vivo models that can be scaled for high-throughput. In this review, we highlight the current status of zebrafish toxicology studies, identify potential future niches for the model in the drug development pipeline, and define the hurdles that must be overcome as zebrafish technologies are refined for systematic toxicology.

Microfluidic device for a rapid immobilization of zebrafish larvae in environmental scanning electron microscopy.

PubMed

Akagi, Jin; Zhu, Feng; Skommer, Joanna; Hall, Chris J; Crosier, Philip S; Cialkowski, Michal; Wlodkowic, Donald

2015-03-01

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. © 2014 International Society for Advancement of Cytometry.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

PubMed

Khan, Kanza M; Collier, Adam D; Meshalkina, Darya A; Kysil, Elana V; Khatsko, Sergey L; Kolesnikova, Tatyana; Morzherin, Yury Yu; Warnick, Jason E; Kalueff, Allan V; Echevarria, David J

2017-07-01

Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets. © 2017 The British Pharmacological Society.

A homeostatic, chip-based platform for zebrafish larvae immobilization and long-term imaging

NASA Astrophysics Data System (ADS)

Friedrich, Timo; Zhu, Feng; Wlodkowic, Donald; Kaslin, Jan

2015-12-01

Zebrafish larvae are ideal for toxicology and drug screens due to their transparency, small size and similarity to humans on the genetic level. Using modern imaging techniques, cells and tissues can be dynamically visualised and followed over days in multiple zebrafish. Yet continued imaging experiments require specialized conditions such as: moisture and heat control to maintain specimen homeostasis. Chambers that control the environment are generally very expensive and are not always available for all imaging platforms. A highly customizable mounting configuration with built-in means of controlling temperature and media flow would therefore be a valuable tool for long term imaging experiments. Rapid prototyping using 3D printing is particularly suitable as a production method as it offers high flexibility in design, is widely available and allows a high degree of customizing. We study neural regeneration in zebrafish. Regeneration is limited in humans, but zebrafish recover from neural damage within days. Yet, the underlying regenerative mechanisms remain unclear. We developed an agarose based mounting system that holds the embryos in defined positions along removable strips. Homeostasis and temperature control is ensured by channels circulating buffer and heated water. This allows to image up to 120 larvae simultaneously for more than two days. Its flexibility and the low-volume, high larvae ratio will allow screening of small compound libraries. Taken together, we offer a low cost, highly adaptable solution for long term in-vivo imaging.

Incorporating zebrafish omics into chemical biology and toxicology.

PubMed

Sukardi, Hendrian; Ung, Choong Yong; Gong, Zhiyuan; Lam, Siew Hong

2010-03-01

In this communication, we describe the general aspects of omics approaches for analyses of transcriptome, proteome, and metabolome, and how they can be strategically incorporated into chemical screening and perturbation studies using the zebrafish system. Pharmacological efficacy and selectivity of chemicals can be evaluated based on chemical-induced phenotypic effects; however, phenotypic observation has limitations in identifying mechanistic action of chemicals. We suggest adapting gene-expression-based high-throughput screening as a complementary strategy to zebrafish-phenotype-based screening for mechanistic insights about the mode of action and toxicity of a chemical, large-scale predictive applications and comparative analysis of chemical-induced omics signatures, which are useful to identify conserved biological responses, signaling pathways, and biomarkers. The potential mechanistic, predictive, and comparative applications of omics approaches can be implemented in the zebrafish system. Examples of these using the omics approaches in zebrafish, including data of ours and others, are presented and discussed. Omics also facilitates the translatability of zebrafish studies across species through comparison of conserved chemical-induced responses. This review is intended to update interested readers with the current omics approaches that have been applied in chemical studies on zebrafish and their potential in enhancing discovery in chemical biology.

Automated feature extraction for retinal vascular biometry in zebrafish using OCT angiography

NASA Astrophysics Data System (ADS)

Bozic, Ivan; Rao, Gopikrishna M.; Desai, Vineet; Tao, Yuankai K.

2017-02-01

Zebrafish have been identified as an ideal model for angiogenesis because of anatomical and functional similarities with other vertebrates. The scale and complexity of zebrafish assays are limited by the need to manually treat and serially screen animals, and recent technological advances have focused on automation and improving throughput. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo imaging of retinal vasculature in zebrafish. OCT-A summed voxel projections were low pass filtered and skeletonized to create an en face vascular map prior to connectivity analysis. Vascular segmentation was referenced to the optic nerve head (ONH), which was identified by automatically segmenting the retinal pigment epithelium boundary on the OCT structural volume. The first vessel branch generation was identified as skeleton segments with branch points closest to the ONH, and subsequent generations were found iteratively by expanding the search space outwards from the ONH. Biometric parameters, including length, curvature, and branch angle of each vessel segment were calculated and grouped by branch generation. Despite manual handling and alignment of each animal over multiple time points, we observe distinct qualitative patterns that enable unique identification of each eye from individual animals. We believe this OCT-based retinal biometry method can be applied for automated animal identification and handling in high-throughput organism-level pharmacological assays and genetic screens. In addition, these extracted features may enable high-resolution quantification of longitudinal vascular changes as a method for studying zebrafish models of retinal neovascularization and vascular remodeling.

Multiple myeloma presenting as CEA-producing rectal cancer

PubMed Central

Talamo, Giampaolo; Barochia, Amitkumar; Zangari, Maurizio; Loughran, Thomas P

2010-01-01

We report the case of a 57-year-old patient with multiple myeloma, characterized by extramedullary involvement of the rectum at presentation. Malignant plasma cells were found to produce carcinoembryonic antigen (CEA), a tumor antigen more commonly associated with rectal adenocarcinomas. PMID:21139949

Combined effects of depleted uranium and ionising radiation on zebrafish embryos.

PubMed

Ng, C Y P; Pereira, S; Cheng, S H; Adam-Guillermin, C; Garnier-Laplace, J; Yu, K N

2015-11-01

In the environment, living organisms are exposed to a mixture of stressors, and the combined effects are deemed as multiple stressor effects. In the present work, the authors studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to alpha particles and depleted uranium (DU) through quantification of apoptotic signals at 24 h post-fertilisation (hpf) revealed by vital dye acridine orange staining. In each set of experiments, dechorionated zebrafish embryos were divided into 4 groups, each having 10 embryos: Group (C) in which the embryos did not receive any further treatment; Group (IU) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and were then exposed to 100 µg l(-1) of DU from 5 to 6 hpf; Group (I) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and Group (U) in which the dechorionated embryos were exposed to 100 µg l(-1) of DU from 5 to 6 hpf. The authors confirmed that an alpha-particle dose of 0.44 mGy and a DU exposure for 1 h separately led to hormetic and toxic effects assessed by counting apoptotic signals, respectively, in the zebrafish. Interestingly, the combined exposure led to an effect more toxic than that caused by the DU exposure alone, so effectively DU changed the beneficial effect (hormesis) brought about by alpha-particle irradiation into an apparently toxic effect. This could be explained in terms of the promotion of early death of cells predisposed to spontaneous transformation by the small alpha-particle dose (i.e. hormetic effect) and the postponement of cell death upon DU exposure. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Prolactin-dependent modulation of organogenesis in the vertebrate: Recent discoveries in zebrafish.

PubMed

Nguyen, Nhu; Stellwag, Edmund J; Zhu, Yong

2008-11-01

The scientific literature is replete with evidence of the multifarious functions of the prolactin (PRL)/growth hormone (GH) superfamily in adult vertebrates. However, little information is available on the roles of PRL and related hormones prior to the adult stage of development. A limited number of studies suggest that GH functions to stimulate glucose transport and protein synthesis in mouse blastocytes and may be involved during mammalian embryogenesis. In contrast, the evidence for a role of PRL during vertebrate embryogenesis is limited and controversial. Genes encoding GH/PRL hormones and their respective receptors are actively transcribed and translated in various animal models at different time points, particularly during tissue remodeling. We have addressed the potential function of GH/PRL hormones during embryonic development in zebrafish by the temporary inhibition of in vivo PRL translation. This treatment caused multiple morphological defects consistent with a role of PRL in embryonic-stage organogenesis. The affected organs and tissues are known targets of PRL activity in fish and homologous structures in mammalian species. Traditionally, the GH/PRL hormones are viewed as classical endocrine hormones, mediating functions through the circulatory system. More recent evidence points to cytokine-like actions of these hormones through either an autocrine or a paracrine mechanism. In some situations they could mimic actions of developmentally regulated genes as suggested by experiments in multiple organisms. In this review, we present similarities and disparities between zebrafish and mammalian models in relation to PRL and PRLR activity. We conclude that the zebrafish could serve as a suitable alternative to the rodent model to study PRL functions in development, especially in relation to organogenesis.

Zebrafish models for the functional genomics of neurogenetic disorders.

PubMed

Kabashi, Edor; Brustein, Edna; Champagne, Nathalie; Drapeau, Pierre

2011-03-01

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Morphologic analysis of the zebrafish digestive system.

PubMed

Trotter, Andrew J; Parslow, Adam C; Heath, Joan K

2009-01-01

The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches-including confocal analysis-to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding number of antibodies that cross-react with zebrafish proteins and the generation of tissue-specific transgenic green fluorescent protein reporter lines that mark specific cell and tissue compartments have greatly enhanced our ability to successfully image the developing zebrafish digestive system.

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

PubMed Central

Lawrence, Christian; Best, Jason; Cockington, Jason; Henry, Eric C.; Hurley, Shane; James, Althea; Lapointe, Christopher; Maloney, Kara; Sanders, Erik

2016-01-01

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding. PMID:26863035

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish.

PubMed

Lawrence, Christian; Best, Jason; Cockington, Jason; Henry, Eric C; Hurley, Shane; James, Althea; Lapointe, Christopher; Maloney, Kara; Sanders, Erik

2016-01-17

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish

PubMed Central

Fang, Longhou; Green, Simone R.; Baek, Ji Sun; Lee, Sang-Hak; Ellett, Felix; Deer, Elena; Lieschke, Graham J.; Witztum, Joseph L.; Tsimikas, Sotirios; Miller, Yury I.

2011-01-01

Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock–induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP–expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans. PMID:22105168

Estrogenic effect of the phytoestrogen biochanin A in zebrafish, Danio rerio, and brown trout, Salmo trutta.

PubMed

Holbech, Henrik; Schröder, Kristoffer D; Nielsen, Marie L; Brande-Lavridsen, Nanna; Holbech, Bente Frost; Bjerregaard, Poul

2013-11-15

Isoflavones with estrogenic activity produced in Fabaceae plants are known to leach from agricultural areas to freshwater systems, but the effect of waterborne isoflavones in fish has not been thoroughly characterized. Therefore, the estrogenic effect of waterborne biochanin A was investigated in zebrafish (Danio rerio) and juvenile brown trout (Salmo trutta). Exposure of juvenile brown trout to 10 μg biochanin AL(-1) or higher caused marked vitellogenin induction after 9-10 days of exposure and so did exposure to 186 μg biochanin AL(-1) for 6h. Following 8d of exposure, a NOEC for induction of vitellogenin production in male zebrafish was 70 and LOEC 114 μg biochanin AL(-1). Exposure to 209 μg biochanin AL(-1) from hatch to 60 days post hatch (dph) caused a skewing of the sex ratio toward more phenotypic female zebrafish, but did not cause induction of vitellogenin in male and undifferentiated fish. (1) biochanin A elicits estrogenic effects in trout at environmentally realistic concentrations, (2) brown trout plasma vitellogenin concentrations respond to lower biochanin A exposure concentrations than vitellogenin concentrations in zebrafish homogenates and (3) concerning vitellogenin induction, the hypothesis should be tested if short term tests with zebrafish may show a higher sensitivity than partial life cycle tests. Copyright © 2013 Elsevier B.V. All rights reserved.

Open-RAC: Open-Design, Recirculating and Auto-Cleaning Zebrafish Maintenance System.

PubMed

Nema, Shubham; Bhargava, Yogesh

2017-08-01

Zebrafish is a vertebrate animal model. Their maintenance in large number under laboratory conditions is a daunting task. Commercially available recirculating zebrafish maintenance systems are used to efficiently handle the tasks of automatic sediment cleaning from zebrafish tanks with minimal waste of water. Due to their compact nature, they also ensure the maximal use of available lab space. However, the high costs of commercial systems present a limitation to researchers with limited funds. A cost-effective zebrafish maintenance system with major features offered by commercially available systems is highly desirable. Here, we describe a compact and recirculating zebrafish maintenance system. Our system is composed of cost-effective components, which are available in local markets and/or can be procured via online vendors. Depending on the expertise of end users, the system can be assembled in 2 days. The system is completely customizable as it offers geometry independent zebrafish tanks that are capable of auto-cleaning the sediments. Due to these features, we called our setup as Open-RAC (Open-design, Recirculating and Auto-Cleaning zebrafish maintenance system). Open-RAC is a cost-effective and viable alternative to the currently available zebrafish maintenance systems. Thus, we believe that the use of Open-RAC could promote the zebrafish research by removing the cost barrier for researchers.

Tools for automating the imaging of zebrafish larvae.

PubMed

Pulak, Rock

2016-03-01

The VAST BioImager system is a set of tools developed for zebrafish researchers who require the collection of images from a large number of 2-7 dpf zebrafish larvae. The VAST BioImager automates larval handling, positioning and orientation tasks. Color images at about 10 μm resolution are collected from the on-board camera of the system. If images of greater resolution and detail are required, this system is mounted on an upright microscope, such as a confocal or fluorescence microscope, to utilize their capabilities. The system loads a larvae, positions it in view of the camera, determines orientation using pattern recognition analysis, and then more precisely positions to user-defined orientation for optimal imaging of any desired tissue or organ system. Multiple images of the same larva can be collected. The specific part of each larva and the desired orientation and position is identified by the researcher and an experiment defining the settings and a series of steps can be saved and repeated for imaging of subsequent larvae. The system captures images, then ejects and loads another larva from either a bulk reservoir, a well of a 96 well plate using the LP Sampler, or individually targeted larvae from a Petri dish or other container using the VAST Pipettor. Alternative manual protocols for handling larvae for image collection are tedious and time consuming. The VAST BioImager automates these steps to allow for greater throughput of assays and screens requiring high-content image collection of zebrafish larvae such as might be used in drug discovery and toxicology studies. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Transvection Arising from Transgene Interactions in Zebrafish.

PubMed

Keefe, Matthew D; Bonkowsky, Joshua L

2017-02-01

There has been a rapid expansion in use of transgenic technologies in zebrafish. We report a novel example of transinteractions of genetic elements, or transvection. This interaction led to a novel expression pattern and illustrates a precautionary example regarding use of transgenes in zebrafish.

Teratological Effects of a Panel of Sixty Water-Soluble Toxicants on Zebrafish Development

PubMed Central

Ali, Shaukat; Aalders, Jeffrey

2014-01-01

Abstract The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay. PMID:24650241

Behavioural fever in zebrafish larvae.

PubMed

Rey, Sonia; Moiche, Visila; Boltaña, Sebastian; Teles, Mariana; MacKenzie, Simon

2017-02-01

Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (T R at 28 °C) and thermal gradient conditions (T CH : 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: T R and T Ch . Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Zebrafish models for translational neuroscience research: from tank to bedside

PubMed Central

Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

2014-01-01

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

Characterization of behavioral and endocrine effects of LSD on zebrafish.

PubMed

Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

2010-12-25

Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

15 years of zebrafish chemical screening

PubMed Central

Rennekamp, Andrew J.; Peterson, Randall T.

2015-01-01

In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies. PMID:25461724

Embryonic Zebrafish Model - A Well-Established Method for Rapidly Assessing the Toxicity of Homeopathic Drugs: - Toxicity Evaluation of Homeopathic Drugs Using Zebrafish Embryo Model.

PubMed

Gupta, Himanshu R; Patil, Yogesh; Singh, Dipty; Thakur, Mansee

2016-12-01

Advancements in nanotechnology have led to nanoparticle (NP) use in various fields of medicine. Although the potential of NPs is promising, the lack of documented evidence on the toxicological effects of NPs is concerning. A few studies have documented that homeopathy uses NPs. Unfortunately, very few sound scientific studies have explored the toxic effects of homeopathic drugs. Citing this lack of high-quality scientific evidence, regulatory agencies have been reluctant to endorse homeopathic treatment as an alternative or adjunct treatment. This study aimed to enhance our insight into the impact of commercially-available homeopathic drugs, to study the presence of NPs in those drugs and any deleterious effects they might have, and to determine the distribution pattern of NPs in zebrafish embryos ( Danio rerio ). Homeopathic dilutions were studied using high-resolution transmission electron microscopy with selected area electron diffraction (SAED). For the toxicity assessment on Zebrafish, embryos were exposed to a test solution from 4 - 6 hours post-fertilization, and embryos/larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. Around 5 dpf was found to be the optimum developmental stage for evaluation. The present study aimed to conclusively prove the presence of NPs in all high dilutions of homeopathic drugs. Embryonic zebrafish were exposed to three homeopathic drugs with two potencies (30CH, 200CH) during early embryogenesis. The resulting morphological and cellular responses were observed. Exposure to these potencies produced no visibly significant malformations, pericardial edema, and mortality and no necrotic and apoptotic cellular death. Our findings clearly demonstrate that no toxic effects were observed for these three homeopathic drugs at the potencies and exposure times used in this study. The embryonic zebrafish

Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

PubMed Central

Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

2009-01-01

Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

The Effect of Chronic Arsenic Exposure in Zebrafish

PubMed Central

Hallauer, Janell; Geng, Xiangrong; Yang, Hung-Chi; Shen, Jian; Tsai, Kan-Jen

2016-01-01

Abstract Arsenic is a prevalent environmental toxin and a Group one human carcinogenic agent. Chronic arsenic exposure has been associated with many human diseases. The aim of this study is to evaluate zebrafish as an animal model to assess arsenic toxicity in elevated long-term arsenic exposure. With prolonged exposure (6 months) to various concentrations of arsenic from 50 ppb to 300 ppb, effects of arsenic accumulation in zebrafish tissues, and phenotypes were investigated. Results showed that there are no significant changes of arsenic retention in zebrafish tissues, and zebrafish did not exhibit any visible tumor formation under arsenic exposure conditions. However, the zebrafish demonstrate a dysfunction in their neurological system, which is reflected by a reduction of locomotive activity. Moreover, elevated levels of the superoxide dismutase (SOD2) protein were detected in the eye and liver, suggesting increased oxidative stress. In addition, the progenies of arsenic-treated parents displayed a smaller biomass (four-fold reduction in body weight) compared with those from their parental controls. This result indicates that arsenic may induce genetic or epigenetic changes that are then passed on to the next generation. Overall, this study demonstrates that zebrafish is a convenient vertebrate model with advantages in the evaluation of arsenic-associated neurological disorders as well as its influences on the offspring. PMID:27140519

Zebrafish sex: a complicated affair

PubMed Central

Liew, Woei Chang

2014-01-01

In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. PMID:24148942

Fusaric acid induces a notochord malformation in zebrafish via copper chelation.

PubMed

Yin, Emily S; Rakhmankulova, Malika; Kucera, Kaury; de Sena Filho, Jose Guedes; Portero, Carolina E; Narváez-Trujillo, Alexandra; Holley, Scott A; Strobel, Scott A

2015-08-01

Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

Zebrafish Models for Human Acute Organophosphorus Poisoning.

PubMed

Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

2015-10-22

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Live imaging of apoptotic cells in zebrafish

PubMed Central

van Ham, Tjakko J.; Mapes, James; Kokel, David; Peterson, Randall T.

2010-01-01

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. PMID:20601526

HCV IRES-Mediated Core Expression in Zebrafish

PubMed Central

Zhang, Jing-Pu; Hu, Zhan-Ying; Tong, Jun-Wei; Ding, Cun-Bao; Peng, Zong-Gen; Zhao, Li-Xun; Song, Dan-Qing; Jiang, Jian-Dong

2013-01-01

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism. PMID:23469178

Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development.

PubMed

Raghunath, Azhwar; Perumal, Ekambaram

2018-01-01

The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

Conserved gene regulation during acute inflammation between zebrafish and mammals

PubMed Central

Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A.

2017-01-01

Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential. PMID:28157230

Zebrafish as a model system to study toxicology.

PubMed

Dai, Yu-Jie; Jia, Yong-Fang; Chen, Na; Bian, Wan-Ping; Li, Qin-Kai; Ma, Yan-Bo; Chen, Yan-Ling; Pei, De-Sheng

2014-01-01

Monitoring and assessing the effects of contaminants in the aquatic eco-environment is critical in protecting human health and the environment. The zebrafish has been widely used as a prominent model organism in different fields because of its small size, low cost, diverse adaptability, short breeding cycle, high fecundity, and transparent embryos. Recent studies have demonstrated that zebrafish sensitivity can aid in monitoring environmental contaminants, especially with the application of transgenic technology in this area. The present review provides a brief overview of recent studies on wild-type and transgenic zebrafish as a model system to monitor toxic heavy metals, endocrine disruptors, and organic pollutants for toxicology. The authors address the new direction of developing high-throughput detection of genetically modified transparent zebrafish to open a new window for monitoring environmental pollutants. © 2013 SETAC.

Screening of Toxic Effects of Bisphenol A and Products of Its Degradation: Zebrafish (Danio rerio) Embryo Test and Molecular Docking.

PubMed

Makarova, Katerina; Siudem, Pawel; Zawada, Katarzyna; Kurkowiak, Justyna

2016-10-01

Bisphenol A (BPA) acts as an endocrine-disrupting compound even at a low concentration. Degradation of BPA could lead to the formation of toxic products. In this study, we compare the toxicity of BPA and seven intermediate products of its degradation. The accuracy of three molecular docking programs (Surflex, Autodock, and Autodock Vina) in predicting the binding affinities of selected compounds to human (ERα, ERβ, and ERRγ) and zebrafish (ERα, ERRγA, and ERRγB) estrogen and estrogen-related receptors was evaluated. The docking experiments showed that 4-isopropylphenol could have similar toxicity to that of BPA due to its high affinity to ERRγ and ERRγB and high octanol-water partitioning coefficient. The least toxic compounds were hydroquinone and phenol. Those compounds as well as BPA were screened in the zebrafish (Danio rerio) embryo test. 4-isopropylphenol had the strongest toxic effect on zebrafish embryos and caused 100% lethality shortly after exposure. BPA caused the delay in development, multiple deformations, and low heartbeats (30 bps), whereas hydroquinone had no impact on the development of the zebrafish embryo. Thus, the results of zebrafish screening are in good agreement with our docking experiment. The molecular docking could be used to screen the toxicity of other xenoestrogens and their products of degradation.

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holowiecki, Andrew

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, mostmore » notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs. - Highlights: • hmox1a, hmox2a, hmox2b and bvrb are sexually dimorphic in expression. • hmox paralogs were induced in adult tissues by cadmium exposure. • hmox1a, hmox1b and bvrb were induced by multiple pro-oxidants zebrafish embryos. • Differential expression of zebrafish hmox paralogs

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christen, Verena; Capelle, Martinus; Fent, Karl, E-mail: karl.fent@fhnw.ch

2013-10-15

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL andmore » Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes.« less

New frontiers for zebrafish management.

PubMed

Lawrence, C

2016-01-01

The zebrafish (Danio rerio) is a preeminent model organism with a wide and expanding utility for numerous scientific disciplines. The same features that once endeared this small freshwater minnow to developmental biologists combined with its relatively high genetic similarity to mammals and the advent of new, more efficient methods for genome editing are now helping to spur expanded growth in its usage in various fields, including toxicology, drug discovery, transplant biology, disease modeling, and even aquaculture. Continued maturation and adoption of the zebrafish model system in these and other fields of science will require that methods and approaches for husbandry and management of these fish in controlled settings be refined and improved to the extent that, ultimately, zebrafish research becomes more reproducible, defined, cost-effective, and accessible to the masses. Knowledge and technology transfer from laboratory animal science and commercial aquaculture will be a necessary part of this development. Copyright © 2016 Elsevier Inc. All rights reserved.

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes.

PubMed

Saad, Moayad; Bijttebier, Sebastiaan; Matheeussen, An; Verbueken, Evy; Pype, Casper; Casteleyn, Christophe; Van Ginneken, Chris; Maes, Louis; Cos, Paul; Van Cruchten, Steven

2018-02-01

This article represents data regarding a study published in Toxicology in vitro entitled " in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds" (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography - accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

Fishing anti(lymph)angiogenic drugs with zebrafish.

PubMed

García-Caballero, Melissa; Quesada, Ana R; Medina, Miguel A; Marí-Beffa, Manuel

2018-02-01

Zebrafish, an amenable small teleost fish with a complex mammal-like circulatory system, is being increasingly used for drug screening and toxicity studies. It combines the biological complexity of in vivo models with a higher-throughput screening capability compared with other available animal models. Externally growing, transparent embryos, displaying well-defined blood and lymphatic vessels, allow the inexpensive, rapid, and automatable evaluation of drug candidates that are able to inhibit neovascularisation. Here, we briefly review zebrafish as a model for the screening of anti(lymph)angiogenic drugs, with emphasis on the advantages and limitations of the different zebrafish-based in vivo assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

A data-driven weighting scheme for multivariate phenotypic endpoints recapitulates zebrafish developmental cascades

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Guozhu, E-mail: gzhang6@ncsu.edu

Zebrafish have become a key alternative model for studying health effects of environmental stressors, partly due to their genetic similarity to humans, fast generation time, and the efficiency of generating high-dimensional systematic data. Studies aiming to characterize adverse health effects in zebrafish typically include several phenotypic measurements (endpoints). While there is a solid biomedical basis for capturing a comprehensive set of endpoints, making summary judgments regarding health effects requires thoughtful integration across endpoints. Here, we introduce a Bayesian method to quantify the informativeness of 17 distinct zebrafish endpoints as a data-driven weighting scheme for a multi-endpoint summary measure, called weightedmore » Aggregate Entropy (wAggE). We implement wAggE using high-throughput screening (HTS) data from zebrafish exposed to five concentrations of all 1060 ToxCast chemicals. Our results show that our empirical weighting scheme provides better performance in terms of the Receiver Operating Characteristic (ROC) curve for identifying significant morphological effects and improves robustness over traditional curve-fitting approaches. From a biological perspective, our results suggest that developmental cascade effects triggered by chemical exposure can be recapitulated by analyzing the relationships among endpoints. Thus, wAggE offers a powerful approach for analysis of multivariate phenotypes that can reveal underlying etiological processes. - Highlights: • Introduced a data-driven weighting scheme for multiple phenotypic endpoints. • Weighted Aggregate Entropy (wAggE) implies differential importance of endpoints. • Endpoint relationships reveal developmental cascade effects triggered by exposure. • wAggE is generalizable to multi-endpoint data of different shapes and scales.« less

Nrf2 and Nrf2-related proteins in development and developmental toxicity: Insights from studies in zebrafish (Danio rerio).

PubMed

Hahn, Mark E; Timme-Laragy, Alicia R; Karchner, Sibel I; Stegeman, John J

2015-11-01

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. Copyright © 2015. Published by Elsevier Inc.

Nrf2 and Nrf2-Related Proteins in Development and Developmental Toxicity: Insights from studies in Zebrafish (Danio rerio)

PubMed Central

Hahn, Mark E.; Timme-Laragy, Alicia R.; Karchner, Sibel I.; Stegeman, John J.

2015-01-01

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. PMID:26130508

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

PubMed

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge.

Effectiveness of Recommended Euthanasia Methods in Larval Zebrafish (Danio rerio)

PubMed Central

Strykowski, Jennifer L; Schech, Joseph M

2015-01-01

The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge. PMID:25651096

Zebrafish xenograft models of cancer and metastasis for drug discovery.

PubMed

Brown, Hannah K; Schiavone, Kristina; Tazzyman, Simon; Heymann, Dominique; Chico, Timothy Ja

2017-04-01

Patients with metastatic cancer suffer the highest rate of cancer-related death, but existing animal models of metastasis have disadvantages that limit our ability to understand this process. The zebrafish is increasingly used for cancer modelling, particularly xenografting of human cancer cell lines, and drug discovery, and may provide novel scientific and therapeutic insights. However, this model system remains underexploited. Areas covered: The authors discuss the advantages and disadvantages of the zebrafish xenograft model for the study of cancer, metastasis and drug discovery. They summarise previous work investigating the metastatic cascade, such as tumour-induced angiogenesis, intravasation, extravasation, dissemination and homing, invasion at secondary sites, assessing metastatic potential and evaluation of cancer stem cells in zebrafish. Expert opinion: The practical advantages of zebrafish for basic biological study and drug discovery are indisputable. However, their ability to sufficiently reproduce and predict the behaviour of human cancer and metastasis remains unproven. For this to be resolved, novel mechanisms must to be discovered in zebrafish that are subsequently validated in humans, and for therapeutic interventions that modulate cancer favourably in zebrafish to successfully translate to human clinical studies. In the meantime, more work is required to establish the most informative methods in zebrafish.

Electroretinogram analysis of the visual response in zebrafish larvae.

PubMed

Chrispell, Jared D; Rebrik, Tatiana I; Weiss, Ellen R

2015-03-16

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

Study on acute toxicity of amoxicillin wastewater to Zebrafish

NASA Astrophysics Data System (ADS)

Xie, Weifang; Shen, Hongyan

2017-12-01

The main research in this paper is to obtain the effect of pharmaceutical wastewater on the acute toxicity of Zebrafish. The experimental method of exposure is used in this research. Experiments were carried out with different groups of pharmaceutical wastewater. Zebrafish was cultivated in a five liter fish tank. In the experiment, according to mortality, initially a 96h preliminary test was carried out at exposure concentrations to determine if the amoxicillin wastewater was toxic and to define the concentration range (24h LC100, 96h LC0) to be employed in the definitive tests. Based on the half lethal concentration of Zebrafish, the acute toxicity of amoxicillin wastewater to Zebrafish was calculated and the toxicity grade of wastewater was determined. In the experiment, the Zebrafish was exposed with amoxicillin wastewater during 96h. The 24h, 48h, 72h and 96h LC50 of amoxicillin wastewater on the Zebrafish were 63.10%, 53.70%, 41.69% and 40.74%, respectively. At 96h, the test time is the longest, and the value of LC50 is the smallest. In the observation period of 96 hours, the LC50 of amoxicillin wastewater were in the range of 40% ~ 60% and the value of Tua is 1 ~ 2. It indicates amoxicillin wastewater is low toxic wastewater when the experimental time is shorter than 48h, amoxicillin wastewater is moderate toxicity wastewater when the experimental time is higher than 48h. According to the experimental data, with the exposure time and the volume percentage of amoxicillin wastewater increases, the mortality rate of Zebrafish is gradually increased and the toxicity of amoxicillin wastewater increases. It indicates that the toxicity of amoxicillin wastewater is the biggest and the effect of wastewater on Zebrafish is greatest. In some ways, the toxicity of amoxicillin wastewater can be affected by the test time.

Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models.

PubMed

Rajan, Vinothkumar; Dellaire, Graham; Berman, Jason N

2016-01-01

The zebrafish is a widely accepted model to study leukemia. The major advantage of studying leukemogenesis in zebrafish is attributed to its short life cycle and superior imaging capacity. This chapter highlights using transgenic- and xenograft-based models in zebrafish to study a specific leukemogenic mutation and analyze therapeutic responses in vivo.

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

PubMed Central

Goody, Michelle; Jurczyszak, Denise; Kim, Carol; Henry, Clarissa

2017-01-01

INTRODUCTION: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. METHODS: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. RESULTS: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. DISCUSSION: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy.

PubMed

Goody, Michelle; Jurczyszak, Denise; Kim, Carol; Henry, Clarissa

2017-10-25

Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune

Myocardial Polyploidization Creates a Barrier to Heart Regeneration in Zebrafish.

PubMed

González-Rosa, Juan Manuel; Sharpe, Michka; Field, Dorothy; Soonpaa, Mark H; Field, Loren J; Burns, Caroline E; Burns, C Geoffrey

2018-02-26

Correlative evidence suggests that polyploidization of heart muscle, which occurs naturally in post-natal mammals, creates a barrier to heart regeneration. Here, we move beyond a correlation by demonstrating that experimental polyploidization of zebrafish cardiomyocytes is sufficient to suppress their proliferative potential during regeneration. Initially, we determined that zebrafish myocardium becomes susceptible to polyploidization upon transient cytokinesis inhibition mediated by dominant-negative Ect2. Using a transgenic strategy, we generated adult animals containing mosaic hearts composed of differentially labeled diploid and polyploid-enriched cardiomyocyte populations. Diploid cardiomyocytes outcompeted their polyploid neighbors in producing regenerated heart muscle. Moreover, hearts composed of equivalent proportions of diploid and polyploid cardiomyocytes failed to regenerate altogether, demonstrating that a critical percentage of diploid cardiomyocytes is required to achieve heart regeneration. Our data identify cardiomyocyte polyploidization as a barrier to heart regeneration and suggest that mobilizing rare diploid cardiomyocytes in the human heart will improve its regenerative capacity. Copyright © 2018 Elsevier Inc. All rights reserved.

Zebrafish Craniofacial Development: A Window into Early Patterning

PubMed Central

Mork, Lindsey; Crump, Gage

2016-01-01

The formation of the face and skull involves a complex series of developmental events mediated by cells derived from the neural crest, endoderm, mesoderm, and ectoderm. Although vertebrates boast an enormous diversity of adult facial morphologies, the fundamental signaling pathways and cellular events that sculpt the nascent craniofacial skeleton in the embryo have proven to be highly conserved from fish to man. The zebrafish Danio rerio, a small freshwater cyprinid fish from eastern India, has served as a popular model of craniofacial development since the 1990s. Unique strengths of the zebrafish model include a simplified skeleton during larval stages, access to rapidly developing embryos for live imaging, and amenability to transgenesis and complex genetics. In this chapter, we describe the anatomy of the zebrafish craniofacial skeleton; its applications as models for the mammalian jaw, middle ear, palate, and cranial sutures; the superior imaging technology available in fish that has provided unprecedented insights into the dynamics of facial morphogenesis; the use of the zebrafish to decipher the genetic underpinnings of craniofacial biology; and finally a glimpse into the most promising future applications of zebrafish craniofacial research. PMID:26589928

Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

PubMed

Arias-Jayo, Nerea; Alonso-Saez, Laura; Ramirez-Garcia, Andoni; Pardo, Miguel A

2018-04-01

The human intestine hosts a vast and complex microbial community that is vital for maintaining several functions related with host health. The processes that determine the gut microbiome composition are poorly understood, being the interaction between species, the external environment, and the relationship with the host the most feasible. Animal models offer the opportunity to understand the interactions between the host and the microbiota. There are different gnotobiotic mice or rat models colonized with the human microbiota, however, to our knowledge, there are no reports on the colonization of germ-free zebrafish with a complex human intestinal microbiota. In the present study, we have successfully colonized 5 days postfertilization germ-free zebrafish larvae with the human intestinal microbiota previously extracted from a donor and analyzed by high-throughput sequencing the composition of the transferred microbial communities that established inside the zebrafish gut. Thus, we describe for first time which human bacteria phylotypes are able to colonize the zebrafish digestive tract. Species with relevant interest because of their linkage to dysbiosis in different human diseases, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Prevotella spp., or Roseburia spp. have been successfully transferred inside the zebrafish digestive tract.

Differential gene expression associated with dietary methylmercury (MeHg) exposure in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio)

PubMed Central

Liu, Qing; Basu, Niladri; Goetz, Giles; Jiang, Nan; Hutz, Reinhold J.; Tonellato, Peter J.; Carvan, Michael J.

2013-01-01

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5 and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at six weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during six weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration. PMID:23529582

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

NASA Astrophysics Data System (ADS)

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-02-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

Comparative Developmental Toxicity and Stress Protein Responses of Dimethyl Sulfoxide to Rare Minnow and Zebrafish Embryos/Larvae.

PubMed

Xiong, Xiaoqin; Luo, Si; Wu, Benli; Wang, Jianwei

2017-02-01

Dimethyl sulfoxide (DMSO), a widely used carrier solvent, can be toxic to test organisms and has species-specific sensitivity. In this study, the developmental toxicity and stress protein responses of DMSO to rare minnow (Gobiocypris rarus) and zebrafish (Danio rerio) with two tests were compared in the early life stage. In the first test, fertilized eggs were exposed to 0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1.0%, 1.5%, and 2.0% v/v of DMSO until 3 days post hatching. In the second test, larvae from 0 to 8 d were exposed to 2% DMSO until 4 days. Our results showed that DMSO was toxic to rare minnow and zebrafish on multiple indexes, and the no-observed-effect concentrations of DMSO in both species were 1.0% and 0.001% for developmental toxicity analysis and stress protein analysis, respectively. Furthermore, rare minnow larvae were more sensitive than zebrafish to DMSO for spinal malformation. The sensitive period for induction of spinal malformation by DMSO was 0-7 d after hatch (dah) for rare minnow and 0-4 dah for zebrafish. Together, these results will provide support to the use of DMSO in ecotoxicological studies using rare minnow and will contribute to a better understanding of the toxicity of DMSO.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

PubMed Central

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-01-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle. PMID:26898711

Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors.

PubMed

Saera-Vila, Alfonso; Louie, Ke'ale W; Sha, Cuilee; Kelly, Ryan M; Kish, Phillip E; Kahana, Alon

2018-01-01

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

Operant models of relapse in zebrafish (Danio rerio): Resurgence, renewal, and reinstatement.

PubMed

Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

2017-09-29

Zebrafish are a widely used animal model in biomedical research, as an alternative to mammals, for having features such as a fully sequenced genome, high fecundity, and low-cost maintenance, but behavioral research with these fish remains scarce. The present study investigated whether zebrafish could be a new animal model for studies on the relapse of behavior (e.g., addiction and overeating) after the behavior has been extinguished. Specifically, we examined whether zebrafish would show three different types of relapse commonly studied with other species: resurgence, renewal, and reinstatement. For resurgence, a target response (i.e., approaching a sensor) was established by presenting a reinforcer (i.e., shrimp eggs) contingent upon the response in Phase 1; the target response was extinguished while introducing reinforcement for an alternative response in Phase 2; neither response produced the reinforcer in Phase 3. For renewal, a target response was established under Context A in Phase 1 and was extinguished under Context B in Phase 2; the fish were placed back in Context A in Phase 3, where extinction remained in effect. For reinstatement, a target response was established in Phase 1 and was extinguished in Phase 2; the reinforcer was presented independently of responding in Phase 3. Each type of relapse occurred in Phase 3. These results replicate and extend previous findings on relapse to a new species and suggest that zebrafish can be a useful animal model for studying the interactions of biological and environmental factors that lead to relapse. Copyright © 2017 Elsevier B.V. All rights reserved.

Method for somatic cell nuclear transfer in zebrafish.

PubMed

Siripattarapravat, Kannika; Cibelli, Jose B

2011-01-01

Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures. Copyright © 2011 Elsevier Inc. All rights reserved.

Anxiogenic-like effects of chronic nicotine exposure in zebrafish.

PubMed

Stewart, Adam Michael; Grossman, Leah; Collier, Adam D; Echevarria, David J; Kalueff, Allan V

2015-12-01

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research. Copyright © 2015 Elsevier Inc. All rights reserved.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights.

PubMed

Harrison, Nicholas R; Laroche, Fabrice J F; Gutierrez, Alejandro; Feng, Hui

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights

PubMed Central

Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

2016-01-01

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

[10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos

PubMed Central

Chen, Huadong; Soroka, Dominique N.; Haider, Jamil; Ferri-Lagneau, Karine F.; Leung, TinChung; Sang, Shengmin

2013-01-01

Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound towards pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos, and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and in humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MSn spectra and comparison to authentic standards that we synthesized. After 24 hours of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and in humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos. PMID:23701129

Zebrafish for the Study of the Biological Effects of Nicotine

PubMed Central

Klee, Eric W.; Schneider, Henning; Hurt, Richard D.; Ekker, Stephen C.

2011-01-01

Introduction: Zebrafish are emerging as a powerful animal model for studying the molecular and physiological effects of nicotine exposure. The zebrafish have many advantageous physical characteristics, including small size, high fecundity rates, and externally developing transparent embryos. When combined with a battery of molecular–genetic tools and behavioral assays, these attributes enable studies to be conducted that are not practical using traditional animal models. Methods: We reviewed the literature on the application of the zebrafish model as a preclinical model to study the biological effects of nicotine exposure. Results: The identified studies used zebrafish to examine the effects of nicotine exposure on early development, addiction, anxiety, and learning. The methods used included green fluorescent protein–labeled proteins to track in vivo nicotine-altered neuron development, nicotine-conditioned place preference, and locomotive sensitization linked with high-throughput molecular and genetic screens and behavioral models of learning and stress response to nicotine. Data are presented on the complete homology of all known human neural nicotinic acetylcholine receptors in zebrafish and on the biological similarity of human and zebrafish dopaminergic signaling. Conclusions: Tobacco dependence remains a major health problem worldwide. Further understanding of the molecular effects of nicotine exposure and genetic contributions to dependence may lead to improvement in patient treatment strategies. While there are limitations to the use of zebrafish as a preclinical model, it should provide a valuable tool to complement existing model systems. The reviewed studies demonstrate the enormous opportunity zebrafish have to advance the science of nicotine and tobacco research. PMID:21385906

In vivo physiological recording from the lateral line of juvenile zebrafish

PubMed Central

Olt, Jennifer; Allen, Claire E.

2016-01-01

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live

A review of monoaminergic neuropsychopharmacology in zebrafish.

PubMed

Maximino, Caio; Herculano, Anderson Manoel

2010-12-01

Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

Effects of calorie restriction on the zebrafish liver proteome

PubMed Central

Jury, David R.; Kaveti, Suma; Duan, Zhong-Hui; Willard, Belinda; Kinter, Michael; Londraville, Richard

2012-01-01

A proteomic approach was taken to study how fish respond to changes in calorie availability, with the longer-term goal of understanding the evolution of lipid metabolism in vertebrates. Zebrafish (Danio rerio) were fed either high (3 rations/day) or low (1 ration/7 days) calorie diets for 5 weeks and liver proteins extracted for proteomic analyses. Proteins were separated on two-dimensional electrophoresis gels and homologous spots compared between treatments to determine which proteins were up-regulated with high-calorie diet. Fifty-five spots were excised from the gel and analyzed via LC–ESI MS/MS, which resulted in the identification of 69 unique proteins (via multiple peptides). Twenty-nine of these proteins were differentially expressed between treatments. Differentially expressed proteins were mapped to Gene Ontology (GO) terms, and these terms compared to the entire zebrafish GO annotation set by Fisher's exact test. The most significant GO terms associated with high-calorie diet are related to a decrease in oxygen-binding activity in the high-calorie treatment. This response is consistent with a well-characterized response in obese humans, indicating there may be a link between lipid storage and hypoxia sensitivity in vertebrates. PMID:20494847

Using zebrafish in systems toxicology for developmental toxicity testing.

PubMed

Nishimura, Yuhei; Inoue, Atsuto; Sasagawa, Shota; Koiwa, Junko; Kawaguchi, Koki; Kawase, Reiko; Maruyama, Toru; Kim, Soonih; Tanaka, Toshio

2016-01-01

With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments. © 2015 Japanese Teratology Society.

Social learning of an associative foraging task in zebrafish

NASA Astrophysics Data System (ADS)

Zala, Sarah M.; Määttänen, Ilmari

2013-05-01

The zebrafish ( Danio rerio) is increasingly becoming an important model species for studies on the genetic and neural mechanisms controlling behaviour and cognition. Here, we utilized a conditioned place preference (CPP) paradigm to study social learning in zebrafish. We tested whether social interactions with conditioned demonstrators enhance the ability of focal naïve individuals to learn an associative foraging task. We found that the presence of conditioned demonstrators improved focal fish foraging behaviour through the process of social transmission, whereas the presence of inexperienced demonstrators interfered with the learning of the control focal fish. Our results indicate that zebrafish use social learning for finding food and that this CPP paradigm is an efficient assay to study social learning and memory in zebrafish.

Tracking zebrafish larvae in group – Status and perspectives☆

PubMed Central

Martineau, Pierre R.; Mourrain, Philippe

2013-01-01

Video processing is increasingly becoming a standard procedure in zebrafish behavior investigations as it enables higher research throughput and new or better measures. This trend, fostered by the ever increasing performance-to-price ratio of the required recording and processing equipment, should be expected to continue in the foreseeable future, with video-processing based methods permeating more and more experiments and, as a result, expanding the very role of behavioral studies in zebrafish research. To assess whether the routine video tracking of zebrafish larvae directly in the Petri dish is a capability that can be expected in the near future, the key processing concepts are discussed and illustrated on published zebrafish studies when available or other animals when not. PMID:23707495

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism

PubMed Central

Spagnoli, Sean; Xue, Lan; Kent, Michael L.

2015-01-01

The zebrafish’s potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host’s brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over ten minutes along with taps at 18 and 60 minutes to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 minutes, whereas exposed negative fish retained partial habituation at 18 minutes. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

PubMed Central

Ohga, Rie; Ota, Satoshi; Kawahara, Atsuo

2015-01-01

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish. PMID:26010089

Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2012-01-01

Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

Cardiac Ca2+ signalling in zebrafish: Translation of findings to man.

PubMed

van Opbergen, Chantal J M; van der Voorn, Stephanie M; Vos, Marc A; de Boer, Teun P; van Veen, Toon A B

2018-05-07

Sudden cardiac death is a leading cause of death worldwide, mainly caused by highly disturbed electrical activation patterns in the heart. Currently, murine models are the most popular model to study underlying molecular mechanisms of inherited or acquired cardiac electrical abnormalities, although the numerous electrophysiological discrepancies between mouse and human raise the question whether mice are the optimal model to study cardiac rhythm disorders. Recently it has been uncovered that the zebrafish cardiac electrophysiology seems surprisingly similar to the human heart, mainly because the zebrafish AP contains a clear plateau phase and ECG characteristics show alignment with the human ECG. Although, before using zebrafish as a model to study cardiac arrhythmogenesis, however, it is very important to gain a better insight into the electrophysiological characteristics of the zebrafish heart. In this review we outline the electrophysiological machinery of the zebrafish cardiomyocytes, with a special focus on the intracellular Ca 2+ dynamics and excitation-contraction coupling. We debate the potential of zebrafish as a model to study human cardiovascular diseases and postulate steps to employ zebrafish into a more 'humanized' model. Copyright © 2018 Elsevier Ltd. All rights reserved.

The ontogeny of sleep-wake cycles in zebrafish: a comparison to humans

PubMed Central

Sorribes, Amanda; Þorsteinsson, Haraldur; Arnardóttir, Hrönn; Jóhannesdóttir, Ingibjörg Þ.; Sigurgeirsson, Benjamín; de Polavieja, Gonzalo G.; Karlsson, Karl Æ.

2013-01-01

Zebrafish (Danio rerio) are used extensively in sleep research; both to further understanding of sleep in general and also as a model of human sleep. To date, sleep studies have been performed in larval and adult zebrafish but no efforts have been made to document the ontogeny of zebrafish sleep–wake cycles. Because sleep differs across phylogeny and ontogeny it is important to validate the use of zebrafish in elucidating the neural substrates of sleep. Here we describe the development of sleep and wake across the zebrafish lifespan and how it compares to humans. We find power-law distributions to best fit wake bout data but demonstrate that exponential distributions, previously used to describe sleep bout distributions, fail to adequately account for the data in either species. Regardless, the data reveal remarkable similarities in the ontogeny of sleep cycles in zebrafish and humans. Moreover, as seen in other organisms, zebrafish sleep levels are highest early in ontogeny and sleep and wake bouts gradually consolidate to form the adult sleep pattern. Finally, sleep percentage, bout duration, bout number, and sleep fragmentation are shown to allow for meaningful comparisons between zebrafish and human sleep. PMID:24312015

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

PubMed

Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

2015-12-01

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

Myomaker mediates fusion of fast myocytes in zebrafish embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles, E-mail: Jean-charles.gabillard@rennes.inra.fr

2014-09-05

Highlights: • Myomaker is transiently expressed in fast myocytes during embryonic myogenesis. • Myomaker is essential for fast myocyte fusion in zebrafish. • The function of myomaker is conserved among Teleostomi. - Abstract: Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they weremore » unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.« less

Development of the zebrafish mesonephros.

PubMed

Diep, Cuong Q; Peng, Zhenzhen; Ukah, Tobechukwu K; Kelly, Paul M; Daigle, Renee V; Davidson, Alan J

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. © 2015 Wiley Periodicals, Inc.

Development of the zebrafish mesonephros

PubMed Central

Diep, Cuong Q.; Peng, Zhenzhen; Ukah, Tobechukwu K.; Kelly, Paul M.; Daigle, Renee V.; Davidson, Alan J.

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. PMID:25677367

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Zebrafish: an important tool for liver disease research.

PubMed

Goessling, Wolfram; Sadler, Kirsten C

2015-11-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Zebrafish: An Important Tool for Liver Disease Research

PubMed Central

Goessling, Wolfram; Sadler, Kirsten C.

2016-01-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. PMID:26319012

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

PubMed

Verma, Ajay D; Parnaik, Veena K

2017-07-01

Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

NASA Astrophysics Data System (ADS)

Huang, Na; Xi, Lei

2016-10-01

Zebrafish is a top vertebrate model to study developmental biology and genetics, and it is becoming increasingly popular for studying human diseases due to its high genome similarity to that of humans and the optical transparency in embryonic stages. However, it becomes difficult for pure optical imaging techniques to volumetric visualize the internal organs and structures of wild-type zebrafish in juvenile and adult stages with excellent resolution and penetration depth. Even with the establishment of mutant lines which remain transparent over the life cycle, it is still a challenge for pure optical imaging modalities to image the whole body of adult zebrafish with micro-scale resolution. However, the method called photoacoustic imaging that combines all the advantages of the optical imaging and ultrasonic imaging provides a new way to image the whole body of the zebrafish. In this work, we developed a non-invasive photoacoustic imaging system with optimized near-infrared illumination and cylindrical scanning to image the zebrafish. The lateral and axial resolution yield to 80 μm and 600 μm, respectively. Multispectral strategy with wavelengths from 690 nm to 930 nm was employed to image various organs inside the zebrafish. From the reconstructed images, most major organs and structures inside the body can be precisely imaged. Quantitative and statistical analysis of absorption for organs under illumination with different wavelengths were carried out.

Ionic channels underlying the ventricular action potential in zebrafish embryo.

PubMed

Alday, Aintzane; Alonso, Hiart; Gallego, Monica; Urrutia, Janire; Letamendia, Ainhoa; Callol, Carles; Casis, Oscar

2014-06-01

Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Behavioral Space of Zebrafish Locomotion and Its Neural Network Analog.

PubMed

Girdhar, Kiran; Gruebele, Martin; Chemla, Yann R

2015-01-01

How simple is the underlying control mechanism for the complex locomotion of vertebrates? We explore this question for the swimming behavior of zebrafish larvae. A parameter-independent method, similar to that used in studies of worms and flies, is applied to analyze swimming movies of fish. The motion itself yields a natural set of fish "eigenshapes" as coordinates, rather than the experimenter imposing a choice of coordinates. Three eigenshape coordinates are sufficient to construct a quantitative "postural space" that captures >96% of the observed zebrafish locomotion. Viewed in postural space, swim bouts are manifested as trajectories consisting of cycles of shapes repeated in succession. To classify behavioral patterns quantitatively and to understand behavioral variations among an ensemble of fish, we construct a "behavioral space" using multi-dimensional scaling (MDS). This method turns each cycle of a trajectory into a single point in behavioral space, and clusters points based on behavioral similarity. Clustering analysis reveals three known behavioral patterns-scoots, turns, rests-but shows that these do not represent discrete states, but rather extremes of a continuum. The behavioral space not only classifies fish by their behavior but also distinguishes fish by age. With the insight into fish behavior from postural space and behavioral space, we construct a two-channel neural network model for fish locomotion, which produces strikingly similar postural space and behavioral space dynamics compared to real zebrafish.

The Behavioral Space of Zebrafish Locomotion and Its Neural Network Analog

PubMed Central

Girdhar, Kiran; Gruebele, Martin; Chemla, Yann R.

2015-01-01

How simple is the underlying control mechanism for the complex locomotion of vertebrates? We explore this question for the swimming behavior of zebrafish larvae. A parameter-independent method, similar to that used in studies of worms and flies, is applied to analyze swimming movies of fish. The motion itself yields a natural set of fish "eigenshapes" as coordinates, rather than the experimenter imposing a choice of coordinates. Three eigenshape coordinates are sufficient to construct a quantitative "postural space" that captures >96% of the observed zebrafish locomotion. Viewed in postural space, swim bouts are manifested as trajectories consisting of cycles of shapes repeated in succession. To classify behavioral patterns quantitatively and to understand behavioral variations among an ensemble of fish, we construct a "behavioral space" using multi-dimensional scaling (MDS). This method turns each cycle of a trajectory into a single point in behavioral space, and clusters points based on behavioral similarity. Clustering analysis reveals three known behavioral patterns—scoots, turns, rests—but shows that these do not represent discrete states, but rather extremes of a continuum. The behavioral space not only classifies fish by their behavior but also distinguishes fish by age. With the insight into fish behavior from postural space and behavioral space, we construct a two-channel neural network model for fish locomotion, which produces strikingly similar postural space and behavioral space dynamics compared to real zebrafish. PMID:26132396

Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

PubMed Central

Ba, Qian; Duan, Juan; Tian, Jia-qiang; Wang, Zi-liang; Chen, Tao; Li, Xiao-guang; Chen, Pei-zhan; Wu, Song-jie; Xiang, Li; Li, Jing-quan; Chu, Rui-ai; Wang, Hui

2013-01-01

Aim: To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms. Methods: The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays. Results: Exposure to DHA (1–10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis. Conclusion: DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA. PMID:23708556

Vitamin D receptor deficiency impairs inner ear development in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Hye-Joo; Biology Department, Princess Nourah University, Riyadh 11671

The biological actions of vitamin D are largely mediated through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, which regulates gene expression in a wide variety of tissues and cells. Mutations in VDR gene have been implicated in ear disorders (hearing loss and balance disorder) but the mechanisms are not well established. In this study, to investigate the role of VDR in inner ear development, morpholino-mediated gene knockdown approaches were used in zebrafish model system. Two paralogs for VDR, vdra and vdrb, have been identified in zebrafish. Knockdown of vdra had no effectmore » on ear development, whereas knockdown of vdrb displayed morphological ear defects including smaller otic vesicles with malformed semicircular canals and abnormal otoliths. Loss-of-vdrb resulted in down-regulation of pre-otic markers, pax8 and pax2a, indicating impairment of otic induction. Furthermore, zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination. These data reveal that VDR signaling plays an important role in ear development. - Highlights: • VDR signaling is involved in ear development. • Knockdown of vdrb causes inner ear malformations during embryogenesis. • Knockdown of vdrb affects otic placode induction. • Knockdown of vdrb reduces the number of sensory hair cells in the inner ear. • Knockdown of vdrb disrupts balance and motor coordination.« less

Microstructured Surface Arrays for Injection of Zebrafish Larvae

PubMed Central

Irimia, Daniel

2017-01-01

Abstract Microinjection of zebrafish larvae is an essential technique for delivery of treatments, dyes, microbes, and xenotransplantation into various tissues. Although a number of casts are available to orient embryos at the single-cell stage, no device has been specifically designed to position hatching-stage larvae for microinjection of different tissues. In this study, we present a reusable silicone device consisting of arrayed microstructures, designed to immobilize 2 days postfertilization larvae in lateral, ventral, and dorsal orientations, while providing maximal access to target sites for microinjection. Injection of rhodamine dextran was used to demonstrate the utility of this device for precise microinjection of multiple anatomical targets. PMID:28151697

Biologically inspired robots elicit a robust fear response in zebrafish

NASA Astrophysics Data System (ADS)

Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

2015-03-01

We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

PubMed

Giarmarco, Michelle M; Cleghorn, Whitney M; Hurley, James B; Brockerhoff, Susan E

2018-05-09

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method's versatility. This protocol was developed for imaging Ca 2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca 2+ or metabolites in Müller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca 2+ , and energy homeostasis.

The neurogenetic frontier--lessons from misbehaving zebrafish.

PubMed

Burgess, Harold A; Granato, Michael

2008-11-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.

Automated image-based phenotypic analysis in zebrafish embryos

PubMed Central

Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

2009-01-01

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Yoon Sun; Jung, Hye Jin; Seok, Seung Hyeok

2013-04-19

Highlights: ► This is the first functional characterization of UQCRB in vivo model. ► Angiogenesis is inhibited with UQCRB loss of function in zebrafish. ► UQCRB is introduced as a prognostic marker for mitochondria- and angiogenesis-related diseases. -- Abstract: As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levelsmore » in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.« less

Evolution of complexity in the zebrafish synapse proteome

PubMed Central

Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

2017-01-01

The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success.

PubMed

Hagedorn, Mary; Carter, Virginia L

2011-01-01

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 10(6) cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 10(6) motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.

Zebrafish Reproduction: Revisiting In Vitro Fertilization to Increase Sperm Cryopreservation Success

PubMed Central

Hagedorn, Mary; Carter, Virginia L.

2011-01-01

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 106 cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 106 motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization. PMID:21698162

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

USGS Publications Warehouse

Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

2009-01-01

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

In vivo physiological recording from the lateral line of juvenile zebrafish.

PubMed

Olt, Jennifer; Allen, Claire E; Marcotti, Walter

2016-10-01

Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More

Zebrafish in Toxicology and Environmental Health.

PubMed

Bambino, Kathryn; Chu, Jaime

2017-01-01

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. © 2017 Elsevier Inc. All rights reserved.

Axonal regeneration in zebrafish spinal cord

PubMed Central

Hui, Subhra Prakash

2018-01-01

Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

Zebrafish in Toxicology and Environmental Health

PubMed Central

Bambino, Kathryn; Chu, Jaime

2018-01-01

As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. PMID:28335863

Axonal regeneration in zebrafish spinal cord.

PubMed

Ghosh, Sukla; Hui, Subhra Prakash

2018-03-01

In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

Quantification of birefringence readily measures the level of muscle damage in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berger, Joachim, E-mail: Joachim.Berger@Monash.edu; Sztal, Tamar; Currie, Peter D.

2012-07-13

Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in anmore » otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.« less

Matrix metalloproteinase-9 plays a role in protecting zebrafish from lethal infection with Listeria monocytogenes by enhancing macrophage migration.

PubMed

Shan, Ying; Zhang, Yikai; Zhuo, Xunhui; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

2016-07-01

Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-μm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

PubMed

Chen, Yi-Fei; Peng, Ju; Fang, Miao; Liu, Yi; Nie, Ling-Hui; Mo, Zhi-Xian; Zhu, Ling-Ling

2016-11-20

To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

PubMed Central

Carroll, Kelli J.; North, Trista E.

2015-01-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

PubMed

Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

2011-01-21

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy.

PubMed

Johnson, Nathan M; Farr, Gist H; Maves, Lisa

2013-09-17

Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.

Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility

PubMed Central

Snell, Kathy; Mittge, Erika; Melancon, Ellie; Montgomery, Rebecca; McFadden, Marcie; Camoriano, Javier; Kent, Michael L.; Whipps, Christopher M.; Peirce, Judy

2016-01-01

Abstract In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities. PMID:27351618

Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, Hayden R.; Radić, Zoran; Taylor, Palmer

The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The k{sub i} values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, k{sub r}, in both zebrafish and human AChE. However, differences between themore » K{sub ox} and k{sub 2} constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, K{sub i} and αK{sub i}, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. - Highlights: • Zebrafish AChE shares significant structural similarities with human AChE. • OP-inhibited zebrafish and human AChE exhibit similar reactivation kinetics. • The zebrafish chorion is permeable to BBB penetrant

Characterization of zebrafish dysferlin by morpholino knockdown

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.

2011-09-23

Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafishmore » dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.« less

Embryotoxic and genotoxic effects of sewage effluents in zebrafish embryo using multiple endpoint testing.

PubMed

Babić, Sanja; Barišić, Josip; Višić, Hrvoje; Sauerborn Klobučar, Roberta; Topić Popović, Natalija; Strunjak-Perović, Ivančica; Čož-Rakovac, Rozelindra; Klobučar, Göran

2017-05-15

Wastewater treatment plant (WWTP) effluents are often complex mixtures of various organic and inorganic substances. Quality control of wastewaters and sludges has been regulated with measuring several physico-chemical parameters and sometimes using biological methods with non-specific responses, while synergistic action mechanisms of contaminants in such complex mixtures is still unknown. Toxic effects of wastewaters within and downstream of the WWTP in City of Virovitica, Croatia, were tested on zebrafish Danio rerio using a set of biomarkers that enabled an insight in wastewaters toxic potential on embryos at the cellular, tissue and the whole organism level during an early ontogenesis (24 and 48 hpf). Exposure of embryos to the wastewater samples from WWTP Virovitica increased mortality and abnormality rate. Heart rate, spontaneous movements and pigmentation formation were also markedly affected. Biochemical markers confirmed the presence of MXR inhibitors in all tested wastewater samples, indicating the increase of pollutant accumulation in the cell/organism. Also, a tendency of DNA damage decrease measured with Comet assay was evident in wastewater samples downstream from WWTP although control levels were not reached in any environmental sample. Histopathological analysis showed that exposure to tested samples resulted in impaired muscle organization, notochord malformation and retardation in eye and brain development at embryos 48 hpf. Furthermore, semi-quantitative histopathology assessment indicated increased percentage of embryo defects in river water sampled several kilometers downstream from the WWTP, confirming toxic potential of WWTP effluents. Extension of the zebrafish embryotoxicity test (ZET) with biochemical and histopathological biomarkers could serve as a guiding principle in biomonitoring of wastewater contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Can Zebrafish be used to Identify Developmentally Neurotoxic Chemicals

EPA Science Inventory

Can Zebrafish be Used to Identify Developmentally Neurotoxic Chemicals? The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental neurotoxicity. We are exploring behavioral methods using zebrafish by desig...

Use of Gnotobiotic Zebrafish to Study Vibrio anguillarum Pathogenicity

PubMed Central

Oyarbide, Usua; Iturria, Iñaki; Rainieri, Sandra

2015-01-01

Abstract We evaluated the use of the gnotobiotic zebrafish system to study the effects of bacterial infection, and analyzed expression of genes involved in zebrafish innate immunity. Using a GFP-labeled strain of Vibrio anguillarum, we fluorescently monitored colonization of the zebrafish intestinal tract and used gene expression analysis to compare changes in genes involved in innate immunity between nongnotobiotic and gnotobiotic larvae. The experiments performed with the gnotobiotic zebrafish reveal new insights into V. anguillarum pathogenesis. Specifically, an alteration of the host immune system was detected through the suppression of a number of innate immune genes (NFKB, IL1B, TLR4, MPX, and TRF) during the first 3 h post infection. This immunomodulation can be indicative of a “stealth mechanism” of mucus invasion in which the pathogen found a sheltered niche, a typical trait of intracellular pathogens. PMID:25548877

Teratogenic potential of antiepileptic drugs in the zebrafish model.

PubMed

Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

2013-01-01

The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data.

The Vital Relationship Between Nutrition and Health in Zebrafish.

PubMed

Watts, Stephen A; Lawrence, Christian; Powell, Mickie; D'Abramo, Louis R

2016-07-01

In the relatively short span of four decades, the zebrafish (Danio rerio) has emerged as an increasingly important model organism for biomedicine and other scientific disciplines. As the scale and sophistication of zebrafish research expands, so too does the need to develop standards that promote the production and maintenance of healthy animals for experiments. A major, but long overlooked, contributor to fish health is nutrition. Historically, feeding practices for laboratory zebrafish have been designed to promote growth and reproductive function. However, as the field matures, it is becoming increasingly clear that the nutritional goals for these animals should evolve beyond basic production to the maintenance of clinically healthy research subjects. This review outlines weaknesses and limitations of current approaches and provides a justification for the development of defined standardized diets that will strengthen and facilitate the continued growth of the zebrafish model system.

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

PubMed

Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

2016-10-07

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Zebrafish Models of Prader-Willi Syndrome: Fast Track to Pharmacotherapeutics

PubMed Central

Spikol, Emma D.; Laverriere, Caroline E.; Robnett, Maya; Carter, Gabriela; Wolfe, Erin; Glasgow, Eric

2016-01-01

Prader-Willi syndrome (PWS) is a rare genetic neurodevelopmental disorder characterized by an insatiable appetite, leading to chronic overeating and obesity. Additional features include short stature, intellectual disability, behavioral problems and incomplete sexual development. Although significant progress has been made in understanding the genetic basis of PWS, the mechanisms underlying the pathogenesis of the disorder remain poorly understood. Treatment for PWS consists mainly of palliative therapies; curative therapies are sorely needed. Zebrafish, Danio rerio, represent a promising way forward for elucidating physiological problems such as obesity and identifying new pharmacotherapeutic options for PWS. Over the last decade, an increased appreciation for the highly conserved biology among vertebrates and the ability to perform high-throughput drug screening has seen an explosion in the use of zebrafish for disease modeling and drug discovery. Here, we review recent advances in developing zebrafish models of human disease. Aspects of zebrafish genetics and physiology that are relevant to PWS will be discussed, and the advantages and disadvantages of zebrafish models will be contrasted with current animal models for this syndrome. Finally, we will present a paradigm for drug screening in zebrafish that is potentially the fastest route for identifying and delivering curative pharmacotherapies to PWS patients. PMID:27857842

Zebrafish heart failure models: opportunities and challenges.

PubMed

Shi, Xingjuan; Chen, Ru; Zhang, Yu; Yun, Junghwa; Brand-Arzamendi, Koroboshka; Liu, Xiangdong; Wen, Xiao-Yan

2018-05-03

Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.

The neurogenetic frontier—lessons from misbehaving zebrafish

PubMed Central

Granato, Michael

2008-01-01

One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish. PMID:18836206

An individual-based model of zebrafish population dynamics accounting for energy dynamics.

PubMed

Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R R

2015-01-01

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level.

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

PubMed Central

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study

PubMed Central

Scerbina, Tanya; Chatterjee, Diptendu; Gerlai, Robert

2012-01-01

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species. PMID:22491827

Evaluation of color preference in zebrafish for learning and memory.

PubMed

Avdesh, Avdesh; Martin-Iverson, Mathew T; Mondal, Alinda; Chen, Mengqi; Askraba, Sreten; Morgan, Newman; Lardelli, Michael; Groth, David M; Verdile, Giuseppe; Martins, Ralph N

2012-01-01

There is growing interest in using zebrafish (Danio rerio) as a model of neurodegenerative disorders such as Alzheimer's disease. A zebrafish model of tauopathies has recently been developed and characterized in terms of presence of the pathological hallmarks (i.e., neurofibrillary tangles and cell death). However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess memory and learning in animal models involve visual stimuli, including color preference. The color preference of zebrafish has received little attention. To validate zebrafish as a model for color-associated-learning and memory, it is necessary to evaluate its natural preferences or any pre-existing biases towards specific colors. In the present study, we have used four different colors (red, yellow, green, and blue) to test natural color preferences of the zebrafish using two procedures: Place preference and T-maze. Results from both experiments indicate a strong aversion toward blue color relative to all other colors (red, yellow, and green) when tested in combinations. No preferences or biases were found among reds, yellows, and greens in the place preference procedure. However, red and green were equally preferred and both were preferred over yellow by zebrafish in the T-maze procedure. The results from the present study show a strong aversion towards blue color compared to red, green, and yellow, with yellow being less preferred relative to red and green. The findings from this study may underpin any further designing of color-based learning and memory paradigms or experiments involving aversion, anxiety, or fear in the zebrafish.

Rapid quantification of neutral lipids and triglycerides during zebrafish embryogenesis.

PubMed

Yoganantharjah, Prusothman; Byreddy, Avinesh R; Fraher, Daniel; Puri, Munish; Gibert, Yann

2017-01-01

The zebrafish is a useful vertebrate model to study lipid metabolism. Oil Red-O (ORO) staining of zebrafish embryos, though sufficient for visualizing the localization of triglycerides, was previously inadequate to quantify neutral lipid abundance. For metabolic studies, it is crucial to be able to quantify lipids during embryogenesis. Currently no cost effective, rapid and reliable method exists to quantify the deposition of neutral lipids and triglycerides. Thin layer chromatography (TLC), gas chromatography and mass spectrometry can be used to accurately measure lipid levels, but are time consuming and costly in their use. Hence, we developed a rapid and reliable method to quantify neutral lipids and triglycerides. Zebrafish embryos were exposed to Rimonabant (Rimo) or WIN 55,212-2 mesylate (WIN), compounds previously shown to modify lipid content during zebrafish embryogenesis. Following this, ORO stain was extracted out of both the zebrafish body and yolk sac and optical density was measured to give an indication of neutral lipid and triglyceride accumulation. Embryos treated with 0.3 microM WIN resulted in increased lipid accumulation, whereas 3 microM Rimo caused a decrease in lipid accumulation during embryogenesis. TLC was performed on zebrafish bodies to validate the developed method. In addition, BODIPY free fatty acids were injected into zebrafish embryos to confirm quantification of changes in lipid content in the embryo. Previously, ORO was limited to qualitative assessment; now ORO can be used as a quantitative tool to directly determine changes in the levels of neutral lipids and triglycerides.

Microcystin-LR affects the hypothalamic-pituitary-inter-renal (HPI) axis in early life stages (embryos and larvae) of zebrafish.

PubMed

Chen, Liang; Wang, Yeke; Giesy, John P; Chen, Feng; Shi, Ting; Chen, Jun; Xie, Ping

2018-05-22

Frequencies and durations of blooms of cyanobacteria are increasing. Some cyanobacteria can produce cyanotoxins including microcystins (MCs). MCs are the most common toxic products of hazardous algal blooms (HABs), with the greatest potential for exposure and to cause toxicity. Recently, MCs have been shown to disrupt endocrine functions. In this study, for the first time, effects of MC-LR on the hypothalamic-pituitary-inter-renal (HPI) axis during early embryonic development (embryos/larvae) of zebrafish (Danio rerio), were investigated. Embryos/larvae of zebrafish were exposed to 1, 10, 100, or 300 μg MC-LR/L during the period of 4-168 h post-fertilization (hpf). Exposure to 300 μg MC-LR/L resulted in significantly greater concentrations of whole-body cortisol than those in controls. Expressions of genes along the HPI axis and mineralocorticoid receptor (MR-) and glucocorticoid receptor (GR-) centered gene networks were evaluated by use of quantitative real-time PCR. Expression of mRNA for crh was significantly down-regulated by exposure to 300 μg MC-LR/L, while expressions of crhbp, crhr1, and crhr2 were significantly up-regulated, relative to controls. MC-LR caused significantly lesser levels of mRNA for steroidogenic genes including hmgra, star, and cyp17, but expression of mRNA for hsd20b was significantly greater than that of controls. Treatment with MC-LR also altered profiles of transcription of MR- and GR-centered gene networks, which might result in multiple responses. Taken together, these results demonstrated that MC-LR affected the corticosteroid-endocrine system of larvae of zebrafish. This study provided valuable insights into molecular mechanisms behind potential toxicity and endocrine disruption of MCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

PubMed

McPartland, J M; Glass, Michelle; Matias, Isabel; Norris, Ryan W; Kilpatrick, C William

2007-05-01

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International

Histone deacetylase 1 is required for the development of the zebrafish inner ear

PubMed Central

He, Yingzi; Tang, Dongmei; Li, Wenyan; Chai, Renjie; Li, Huawei

2016-01-01

Histone deacetylase 1 (HDAC1) has been reported to be important for multiple aspects of normal embryonic development, but little is known about its function in the development of mechanosensory organs. Here, we first confirmed that HDAC1 is expressed in the developing otic vesicles of zebrafish by whole-mount in situ hybridization. Knockdown of HDAC1 using antisense morpholino oligonucleotides in zebrafish embryos induced smaller otic vesicles, abnormal otoliths, malformed or absent semicircular canals, and fewer sensory hair cells. HDAC1 loss of function also caused attenuated expression of a subset of key genes required for otic vesicle formation during development. Morpholino-mediated knockdown of HDAC1 resulted in decreased expression of members of the Fgf family in the otic vesicles, suggesting that HDAC1 is involved in the development of the inner ear through regulation of Fgf signaling pathways. Taken together, our results indicate that HDAC1 plays an important role in otic vesicle formation. PMID:26832938

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

PubMed Central

Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

2016-01-01

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. PMID:27241320

Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

NASA Astrophysics Data System (ADS)

Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

2016-05-01

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration.

Zebrafish AID is capable of deaminating methylated deoxycytidines

PubMed Central

Abdouni, Hala; King, Justin J.; Suliman, Mussa; Quinlan, Matthew; Fifield, Heather; Larijani, Mani

2013-01-01

Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure. PMID:23585279

Embryonic senescence and laminopathies in a progeroid zebrafish model.

PubMed

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

2011-03-30

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with

Reproductive toxicity of azoxystrobin to adult zebrafish (Danio rerio).

PubMed

Cao, Fangjie; Zhu, Lizhen; Li, Hui; Yu, Song; Wang, Chengju; Qiu, Lihong

2016-12-01

In the past few decades, extensive application of azoxystrobin has led to great concern regarding its adverse effects on aquatic organisms. The objective of the present study was to evaluate the reproductive toxicity of azoxystrobin to zebrafish. After adult zebrafish of both sexes were exposed to 2, 20 and 200 μg/L azoxystrobin for 21 days, egg production, the fertilization rate, the gonadosomatic index (GSI) and hepatosomatic index (HSI), 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) concentrations, and histological alterations in the gonads and livers were measured. Meanwhile, expression alterations of genes encoding gonadotropins and gonadotropin receptors (fshb, lhb, fshr and lhr), steroid hormone receptors (era, er2b and ar), steroidogenic enzymes (cyp11a, cyp11b, cyp17, cyp19a, cyp19b, hsd3b and hsd17b) in the hypothalamic-pituitary-gonad (HPG) axis and vitellogenin (vtg1 and vtg2) in the livers were also investigated. The results showed that reduced egg production and fertilization rates were observed at 200 μg/L azoxystrobin. In female zebrafish, reduced E2 and Vtg concentrations, decreased GSI, increased T concentrations, and histological alterations in the ovaries and livers were observed at 200 μg/L azoxystrobin, along with significant down-regulation of lhb, cyp19b, lhr, cyp19a, vtg1 and vtg2, and up-regulation of cyp17, hsd3b and hsd17b. In male zebrafish, increased E2 and Vtg concentrations, reduced T concentration and GSI, and histological alterations in the testes and livers were observed after exposure to 20 and 200 μg/L azoxystrobin, along with significant up-regulations of cyp19b, cyp11a, cyp17, cyp19a, hsd3b and hsd17b, vtg1 and vtg2. Moreover, cyp11a, hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish were significantly up-regulated after treatment with 2 μg/L azoxystrobin. The results of the present study indicate that azoxystrobin led to reproductive toxicity in zebrafish and male zebrafish were more sensitive to

Methods for studying the zebrafish brain: past, present and future.

PubMed

Wyatt, Cameron; Bartoszek, Ewelina M; Yaksi, Emre

2015-07-01

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System

PubMed Central

Bugel, Sean M.; Bonventre, Josephine A.; Tanguay, Robert L.

2016-01-01

Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay—an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1–50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization. PMID:27492224

Optimizing multi-dimensional high throughput screening using zebrafish

PubMed Central

Truong, Lisa; Bugel, Sean M.; Chlebowski, Anna; Usenko, Crystal Y.; Simonich, Michael T.; Massey Simonich, Staci L.; Tanguay, Robert L.

2016-01-01

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. PMID:27453428

Behavioral effects of MDMA ('ecstasy') on adult zebrafish.

PubMed

Stewart, Adam; Riehl, Russell; Wong, Keith; Green, Jeremy; Cosgrove, Jessica; Vollmer, Karoly; Kyzar, Evan; Hart, Peter; Allain, Alexander; Cachat, Jonathan; Gaikwad, Siddharth; Hook, Molly; Rhymes, Kate; Newman, Alan; Utterback, Eli; Chang, Katie; Kalueff, Allan V

2011-06-01

3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is a potent psychedelic drug inducing euphoria and hypersociability in humans, as well as hyperactivity and anxiety in rodents. Adult zebrafish (Danio rerio) have become a widely used species in neurobehavioral research. Here, we explore the effects of a wide range (0.25-120 mg/l) of acute MDMA doses on zebrafish behavior in the novel tank test. Although MDMA was inactive at lower doses (0.25-10 mg/l), higher doses reduced bottom swimming and immobility (40-120 mg/l) and impaired intrasession habituation (10-120 mg/l). MDMA also elevated brain c-fos expression, collectively confirming the usage of zebrafish models for screening of hallucinogenic compounds.

An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

PubMed Central

Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R. R.

2015-01-01

Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level. PMID:25938409

Circadian clock regulation of the cell cycle in the zebrafish intestine.

PubMed

Peyric, Elodie; Moore, Helen A; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.

Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

PubMed Central

Peyric, Elodie; Moore, Helen A.; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally. PMID:24013905

Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development.

PubMed

Hen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D; Yaniv, Karina

2015-12-15

Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds. © 2015. Published by The Company of Biologists Ltd.

Behaviour of telomere and telomerase during aging and regeneration in zebrafish.

PubMed

Anchelin, Monique; Murcia, Laura; Alcaraz-Pérez, Francisca; García-Navarro, Esther M; Cayuela, María L

2011-02-09

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.

The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos

PubMed Central

2012-01-01

Background Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome. Methods Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group. Results The attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2). Conclusions Based on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner. PMID:22776023

Teratogenic Potential of Antiepileptic Drugs in the Zebrafish Model

PubMed Central

Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

2013-01-01

The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data. PMID:24324971

Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

PubMed Central

Elsen, Gina E.; Choi, Louis; Millen, Kathleen; Grinblat, Yevgenya; Prince, Victoria E.

2008-01-01

During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain. PMID:18191121

Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

PubMed

Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

2015-12-01

Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

Automated phenotype pattern recognition of zebrafish for high-throughput screening.

PubMed

Schutera, Mark; Dickmeis, Thomas; Mione, Marina; Peravali, Ravindra; Marcato, Daniel; Reischl, Markus; Mikut, Ralf; Pylatiuk, Christian

2016-07-03

Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.

Midline signals regulate retinal neurogenesis in zebrafish.

PubMed

Masai, I; Stemple, D L; Okamoto, H; Wilson, S W

2000-08-01

In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.

Using Zebrafish to Study Podocyte Genesis During Kidney Development and Regeneration

PubMed Central

Kroeger, Paul T.; Wingert, Rebecca A.

2014-01-01

SUMMARY During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. PMID:24920186

Functional Development of the Circadian Clock in the Zebrafish Pineal Gland

PubMed Central

Ben-Moshe, Zohar; Foulkes, Nicholas S.

2014-01-01

The zebrafish constitutes a powerful model organism with unique advantages for investigating the vertebrate circadian timing system and its regulation by light. In particular, the remarkably early and rapid development of the zebrafish circadian system has facilitated exploring the factors that control the onset of circadian clock function during embryogenesis. Here, we review our understanding of the molecular basis underlying functional development of the central clock in the zebrafish pineal gland. Furthermore, we examine how the directly light-entrainable clocks in zebrafish cell lines have facilitated unravelling the general mechanisms underlying light-induced clock gene expression. Finally, we summarize how analysis of the light-induced transcriptome and miRNome of the zebrafish pineal gland has provided insight into the regulation of the circadian system by light, including the involvement of microRNAs in shaping the kinetics of light- and clock-regulated mRNA expression. The relative contributions of the pineal gland central clock and the distributed peripheral oscillators to the synchronization of circadian rhythms at the whole animal level are a crucial question that still remains to be elucidated in the zebrafish model. PMID:24839600

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

PubMed

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-04-13

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

PubMed Central

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-01-01

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

PubMed

Radev, Zlatko; Hermel, Jean-Michel; Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

PubMed Central

Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

2015-01-01

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders. PMID:26221953

Model of voluntary ethanol intake in zebrafish: Effect on behavior and hypothalamic orexigenic peptides

PubMed Central

Sterling, M.E.; Karatayev, O.; Chang, G.-Q.; Algava, D.B.; Leibowitz, S.F

2014-01-01

Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period of time to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically-relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake. PMID:25257106

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio).

PubMed

Cao, Fangjie; Wu, Peizhuo; Huang, Lan; Li, Hui; Qian, Le; Pang, Sen; Qiu, Lihong

2018-05-01

Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by

Preparing for a Semiannual IACUC Inspection of a Satellite Zebrafish (Danio rerio) Facility

PubMed Central

Koerber, Amy S; Kalishman, Jennifer

2009-01-01

Institutions worldwide have experienced a rapid growth in the use of zebrafish as a research model for a variety of molecular and genetic studies of vertebrate development. This expansion in zebrafish research essentially has outpaced the establishment of specific recommendations for the care and use of fish in research. In some cases, this situation has created a dilemma where an Institutional Animal Care and Use Committee, which is responsible for oversight of vertebrate animal research, is not fully prepared to undertake this role for a decentralized zebrafish facility. IACUC inspectors will be more equipped to ask pertinent questions by understanding the basic principles of zebrafish health and facility management. Concurrently, zebrafish facility managers can contribute to the progress of a semiannual facility inspection by maintaining fully accessible operating records. In the context of presenting a well-established and useful model of zebrafish management and recordkeeping to the zebrafish facility operator, the information we present here also prepares a potential IACUC inspector to conduct a constructive and positive inspection. PMID:19245754

Multiple functions of GABA A and GABA B receptors during pattern processing in the zebrafish olfactory bulb.

PubMed

Tabor, Rico; Yaksi, Emre; Friedrich, Rainer W

2008-07-01

gamma-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABA(A) and GABA(B) receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABA(B) receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABA(B) receptors have a modulatory influence on OB output activity. The blockade of GABA(A) receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABA(A) receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABA(A) and GABA(B) receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB.

DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

EPA Science Inventory

As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish pigmentation.

PubMed

Le, Hoa Thi; Hong, Bin Na; Lee, Yeong Ro; Cheon, Ji Hyun; Kang, Tong Ho; Kim, Tae Woo

2016-01-15

We synthesized two hydroquinone-tetraethylene glycol conjugates (HQ-TGs) and investigated their logP, photophysical stability, and redox chemical stability. HQ-TGs are a little more hydrophilic than hydroquinone (HQ) and show an enhanced photophysical and redox chemical stability compared with HQ. In addition we studied the effect of HQ-TGs on cell viability and on zebrafish pigmentation. MTT assay in HF-16 cells showed HQ-TGs are less cytotoxic than HQ. The phenotype-based image analysis of zebrafish larvae suggests that HQ-TGs suppress the pigmentation of zebrafish in a dose-dependent manner. The comparative experiments on stability, cytotoxicity, and zebrafish pigmentation between HQ and HQ-TGs suggest that mono tetraethylene glycol-functionalization of HQ is an alternative solution to overcome the adverse effect of HQ. Copyright © 2015 Elsevier Ltd. All rights reserved.

Can zebrafish be used as animal model to study Alzheimer's disease?

PubMed Central

Santana, Soraya; Rico, Eduardo P; Burgos, Javier S

2012-01-01

Zebrafish is rapidly emerging as a promising model organism to study various central nervous system (CNS) disorders, including Alzheimer’s disease (AD). AD is the main cause of dementia in the human population and there is an urgency to understand the causes of this neurodegenerative disease. In this respect, the development of new animal models to study the underlying neurodegenerative mechanisms of AD is an urgent need. In this review we analyze the current situation in the use of zebrafish as a model for AD, discussing the reasons to use this experimental paradigm in CNS investigation and analyzing the several strategies adopted to induce an AD-like pathology in zebrafish. We discuss the strategies of performing interventions to cause damage in the zebrafish brain by altering the major neurotransmitter systems (such as cholinergic, glutamatergic or GABAergic circuits). We also analyze the several transgenic zebrafish constructed for the AD study, discussing both the familial-AD models based on APP processing pathway (APP and presenilins) and in the TAU hyperphosphorylation, together with the genes involved in sporadic-AD, as apolipoprotein E. We conclude that zebrafish is in a preliminary stage of development in the AD field, and that the transgenic animals must be improved to use this fish as an optimal model for AD research. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of the injury caused by alterations in the major neurotransmitter systems are needed. PMID:23383380

Correlation between photoreceptor injury-regeneration and behavior in a zebrafish model.

PubMed

Wang, Ya-Jie; Cai, Shi-Jiao; Cui, Jian-Lin; Chen, Yang; Tang, Xin; Li, Yu-Hao

2017-05-01

Direct exposure to intensive visible light can lead to solar retinopathy, including macular injury. The signs and symptoms include central scotoma, metamorphopsia, and decreased vision. However, there have been few studies examining retinal injury due to intensive light stimulation at the cellular level. Neural network arrangements and gene expression patterns in zebrafish photoreceptors are similar to those observed in humans, and photoreceptor injury in zebrafish can induce stem cell-based cellular regeneration. Therefore, the zebrafish retina is considered a useful model for studying photoreceptor injury in humans. In the current study, the central retinal photoreceptors of zebrafish were selectively ablated by stimulation with high-intensity light. Retinal injury, cell proliferation and regeneration of cones and rods were assessed at 1, 3 and 7 days post lesion with immunohistochemistry and in situ hybridization. Additionally, a light/dark box test was used to assess zebrafish behavior. The results revealed that photoreceptors were regenerated by 7 days after the light-induced injury. However, the regenerated cells showed a disrupted arrangement at the lesion site. During the injury-regeneration process, the zebrafish exhibited reduced locomotor capacity, weakened phototaxis and increased movement angular velocity. These behaviors matched the morphological changes of retinal injury and regeneration in a number of ways. This study demonstrates that the zebrafish retina has a robust capacity for regeneration. Visual impairment and stress responses following high-intensity light stimulation appear to contribute to the alteration of behaviors.

Evidence for a core gut microbiota in the zebrafish

PubMed Central

Roeselers, Guus; Mittge, Erika K; Stephens, W Zac; Parichy, David M; Cavanaugh, Colleen M; Guillemin, Karen; Rawls, John F

2011-01-01

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status. PMID:21472014

Zebrafish models of human eye and inner ear diseases.

PubMed

Blanco-Sánchez, B; Clément, A; Phillips, J B; Westerfield, M

2017-01-01

Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

A bioenergetic model for zebrafish Danio rerio (Hamilton)

USGS Publications Warehouse

Chizinski, C.J.; Sharma, Bibek; Pope, K.L.; Patino, R.

2008-01-01

A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18-32?? C) of water temperatures, and evaluated with a 50 day laboratory trial at 28?? C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass. ?? 2008 The Authors.

Optimizing multi-dimensional high throughput screening using zebrafish.

PubMed

Truong, Lisa; Bugel, Sean M; Chlebowski, Anna; Usenko, Crystal Y; Simonich, Michael T; Simonich, Staci L Massey; Tanguay, Robert L

2016-10-01

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

PubMed

Seto, Sai-Wang; Kiat, Hosen; Lee, Simon M Y; Bensoussan, Alan; Sun, Yu-Ting; Hoi, Maggie P M; Chang, Dennis

2015-12-05

The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

In-silico experiments of zebrafish behaviour: modeling swimming in three dimensions

NASA Astrophysics Data System (ADS)

Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2017-01-01

Zebrafish is fast becoming a species of choice in biomedical research for the investigation of functional and dysfunctional processes coupled with their genetic and pharmacological modulation. As with mammals, experimentation with zebrafish constitutes a complicated ethical issue that calls for the exploration of alternative testing methods to reduce the number of subjects, refine experimental designs, and replace live animals. Inspired by the demonstrated advantages of computational studies in other life science domains, we establish an authentic data-driven modelling framework to simulate zebrafish swimming in three dimensions. The model encapsulates burst-and-coast swimming style, speed modulation, and wall interaction, laying the foundations for in-silico experiments of zebrafish behaviour. Through computational studies, we demonstrate the ability of the model to replicate common ethological observables such as speed and spatial preference, and anticipate experimental observations on the correlation between tank dimensions on zebrafish behaviour. Reaching to other experimental paradigms, our framework is expected to contribute to a reduction in animal use and suffering.

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

PubMed Central

Motaln, Helena; Turnšek, Tamara Lah

2015-01-01

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma. PMID:26109632

In-silico experiments of zebrafish behaviour: modeling swimming in three dimensions

PubMed Central

Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2017-01-01

Zebrafish is fast becoming a species of choice in biomedical research for the investigation of functional and dysfunctional processes coupled with their genetic and pharmacological modulation. As with mammals, experimentation with zebrafish constitutes a complicated ethical issue that calls for the exploration of alternative testing methods to reduce the number of subjects, refine experimental designs, and replace live animals. Inspired by the demonstrated advantages of computational studies in other life science domains, we establish an authentic data-driven modelling framework to simulate zebrafish swimming in three dimensions. The model encapsulates burst-and-coast swimming style, speed modulation, and wall interaction, laying the foundations for in-silico experiments of zebrafish behaviour. Through computational studies, we demonstrate the ability of the model to replicate common ethological observables such as speed and spatial preference, and anticipate experimental observations on the correlation between tank dimensions on zebrafish behaviour. Reaching to other experimental paradigms, our framework is expected to contribute to a reduction in animal use and suffering. PMID:28071731

Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

PubMed Central

Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

2013-01-01

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine

The photoreceptive cells of the pineal gland in adult zebrafish (Danio rerio).

PubMed

Laurà, Rosaria; Magnoli, Domenico; Zichichi, Rosalia; Guerrera, Maria Cristina; De Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

2012-03-01

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions. Copyright © 2011 Wiley Periodicals, Inc.

Increased radial glia quiescence, decreased reactivation upon injury and unaltered neuroblast behavior underlie decreased neurogenesis in the aging zebrafish telencephalon.

PubMed

Edelmann, Kathrin; Glashauser, Lena; Sprungala, Susanne; Hesl, Birgit; Fritschle, Maike; Ninkovic, Jovica; Godinho, Leanne; Chapouton, Prisca

2013-09-01

The zebrafish has recently become a source of new data on the mechanisms of neural stem cell (NSC) maintenance and ongoing neurogenesis in adult brains. In this vertebrate, neurogenesis occurs at high levels in all ventricular regions of the brain, and brain injuries recover successfully, owing to the recruitment of radial glia, which function as NSCs. This new vertebrate model of adult neurogenesis is thus advancing our knowledge of the molecular cues in use for the activation of NSCs and fate of their progeny. Because the regenerative potential of somatic stem cells generally weakens with increasing age, it is important to assess the extent to which zebrafish NSC potential decreases or remains unaltered with age. We found that neurogenesis in the ventricular zone, in the olfactory bulb, and in a newly identified parenchymal zone of the telencephalon indeed declines as the fish ages and that oligodendrogenesis also declines. In the ventricular zone, the radial glial cell population remains largely unaltered morphologically but enters less frequently into the cell cycle and hence produces fewer neuroblasts. The neuroblasts themselves do not change their behavior with age and produce the same number of postmitotic neurons. Thus, decreased neurogenesis in the physiologically aging zebrafish brain is correlated with an increasing quiescence of radial glia. After injuries, radial glia in aged brains are reactivated, and the percentage of cell cycle entry is increased in the radial glia population. However, this reaction is far less pronounced than in younger animals, pointing to irreversible changes in aging zebrafish radial glia. Copyright © 2013 Wiley Periodicals, Inc.

Graph theoretical model of a sensorimotor connectome in zebrafish.

PubMed

Stobb, Michael; Peterson, Joshua M; Mazzag, Borbala; Gahtan, Ethan

2012-01-01

Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome.

Expression of sall4 in taste buds of zebrafish.

PubMed

Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

2013-07-01

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

Zebrafish Discoveries in Cancer Epigenetics.

PubMed

Chernyavskaya, Yelena; Kent, Brandon; Sadler, Kirsten C

2016-01-01

The cancer epigenome is fundamentally different than that of normal cells. How these differences arise in and contribute to carcinogenesis is not known, and studies using model organisms such as zebrafish provide an opportunity to address these important questions. Modifications of histones and DNA comprise the complex epigenome, and these influence chromatin structure, genome stability and gene expression, all of which are fundamental to the cellular changes that cause cancer. The cancer genome atlas covers the wide spectrum of genetic changes associated with nearly every cancer type, however, this catalog is currently uni-dimensional. As the pattern of epigenetic marks and chromatin structure in cancer cells is described and overlaid on the mutational landscape, the map of the cancer genome becomes multi-dimensional and highly complex. Two major questions remain in the field: (1) how the epigenome becomes repatterned in cancer and (2) which of these changes are cancer-causing. Zebrafish provide a tractable in vivo system to monitor the epigenome during transformation and to identify epigenetic drivers of cancer. In this chapter, we review principles of cancer epigenetics and discuss recent work using zebrafish whereby epigenetic modifiers were established as cancer driver genes, thus providing novel insights into the mechanisms of epigenetic reprogramming in cancer.

Zebrafish antipredatory responses: A future for translational research?

PubMed Central

Gerlai, Robert

2011-01-01

Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

Egfl6 is involved in zebrafish notochord development.

PubMed

Wang, Xueqian; Wang, Xin; Yuan, Wei; Chai, Renjie; Liu, Dong

2015-08-01

The epidermal growth factor (EGF) repeat motif defines a superfamily of diverse protein involved in regulating a variety of cellular and physiological processes, such as cell cycle, cell adhesion, proliferation, migration, and neural development. Egfl6, an EGF protein, also named MAGE was first cloned in human tissue. Up to date, the study of zebrafish Egfl6 expression pattern and functional analysis of Egfl6 involved in embryonic development of vertebrate in vivo is thus far lacking. Here we reported that Egfl6 was involved in zebrafish notochord development. It was shown that Egfl6 mRNA was expressed in zebrafish, developing somites, fin epidermis, pharyngeal arches, and hindbrain region. Particularly the secreted Egfl6 protein was significantly accumulated in notochord. Loss of Egfl6 function in zebrafish embryos resulted in curved body with distorted notochord in the posterior trunk. It was observed that expression of all Notch ligand and receptors in notochord of 28 hpf Egfl6 morphants was not affected, except notch2, which was up-regulated. We found that inhibition of Notch signaling by DAPT efficiently rescued notochord developmental defect of Egfl6 deficiency embryos.

Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

PubMed Central

Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

2014-01-01

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

PubMed

Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils

2017-12-01

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

The Visual System of Zebrafish and its Use to Model Human Ocular Diseases

PubMed Central

Gestri, Gaia; Link, Brian A; Neuhauss, Stephan CF

2011-01-01

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually-driven behaviors in the newly hatched larvae. The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases. Here, we review the anatomy, physiology and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases. PMID:21595048

Zebrafish Development: High-throughput Test Systems to Assess Developmental Toxicity

EPA Science Inventory

Abstract Because of its developmental concordance, ease of handling and rapid development, the small teleost, zebrafish (Danio rerio), is frequently promoted as a vertebrate model for medium-throughput developmental screens. This present chapter discusses zebrafish as an altern...

Retinoid regulation of the zebrafish cyp26a1 promoter.

PubMed

Hu, Ping; Tian, Miao; Bao, Jie; Xing, Guangdong; Gu, Xingxing; Gao, Xiang; Linney, Elwood; Zhao, Qingshun

2008-12-01

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533-base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA. (c) 2008 Wiley-Liss, Inc.

Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.

PubMed

Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C; Connors, Lawreen H; Merlini, Giampaolo; Falk, Rodney H; MacRae, Calum A; Liao, Ronglih

2013-07-01

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

Zebrafish Locomotor Responses Predict Irritant Potential of ...

EPA Pesticide Factsheets

Over the past few decades, the drying and warming trends of global climate change have increased wildland fire (WF) season length, as well as geographic area impacted. Consequently, exposures to WF fine particulate matter (PM2.5; aerodynamic diameter <2.5 µm) are likely to increase in frequency and duration, contributing to a growing public health burden. Given the influence of fuel type and combustion conditions on WFPM2.5 composition, there is pressing need to identify the biomass fuel sources and emission constituents that drive toxicity. Previously, we reported the utility of 6-day post-fertilization (dpf) zebrafish larvae in evaluating diesel exhaust PM-induced irritation, demonstrating responses analogous to those in mammals. In the present study, combustions, separated by smoldering or flaming conditions, of pine needles, red oak, pine, eucalyptus, and peat were achieved using an automated tube furnace paired with a cryo-trapping apparatus to collect condensates of emissions. The condensates were extracted and prepared for use in zebrafish assays. We hypothesized that 1) the extractable organic fractions of biomass smoke PM will elicit dose-dependent irritant responses in 6-dpf zebrafish larvae, and 2) the relative potencies will vary across biomass emissions, potentially driven by varying chemical composition of fuel sources. Six-dpf zebrafish (n= 28-32/group) were exposed acutely to PM extracts (5 concentrations; 0.3-30 µg/ml; half-log intervals) and

A two-scale model for correlation between B cell VDJ usage in zebrafish

NASA Astrophysics Data System (ADS)

Pan, Keyao; Deem, Michael

2011-03-01

The zebrafish (Danio rerio) is one of the model animals for study of immunology. The dynamics of the adaptive immune system in zebrafish is similar to that in higher animals. In this work, we built a two-scale model to simulate the dynamics of B cells in primary and secondary immune reactions in zebrafish and to explain the reported correlation between VDJ usage of B cell repertoires in distinct zebrafish. The first scale of the model consists of a generalized NK model to simulate the B cell maturation process in the 10-day primary immune response. The second scale uses a delay ordinary differential equation system to model the immune responses in the 6-month lifespan of zebrafish. The generalized NK model shows that mature B cells specific to one antigen mostly possess a single VDJ recombination. The probability that mature B cells in two zebrafish have the same VDJ recombination increases with the B cell population size or the B cell selection intensity and decreases with the B cell hypermutation rate. The ODE model shows a distribution of correlation in the VDJ usage of the B cell repertoires in two six-month-old zebrafish that is highly similar to that from experiment. This work presents a simple theory to explain the experimentally observed correlation in VDJ usage of distinct zebrafish B cell repertoires after an immune response.

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test.

PubMed

Hallare, Arnold V; Ruiz, Paulo Lorenzo S; Cariño, J C Earl D

2014-05-01

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Textile dyes induce toxicity on zebrafish early life stages.

PubMed

de Oliveira, Gisele Augusto Rodrigues; de Lapuente, Joaquín; Teixidó, Elisabet; Porredón, Constança; Borràs, Miquel; de Oliveira, Danielle Palma

2016-02-01

Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment. © 2015 SETAC.

Understanding spatio-temporal strategies of adult zebrafish exploration in the open field test.

PubMed

Stewart, Adam Michael; Gaikwad, Siddharth; Kyzar, Evan; Kalueff, Allan V

2012-04-27

Zebrafish (Danio rerio) are emerging as a useful model organism for neuroscience research. Mounting evidence suggests that various traditional rodent paradigms may be adapted for testing zebrafish behavior. The open field test is a popular rodent test of novelty exploration, recently applied to zebrafish research. To better understand fish novelty behavior, we exposed adult zebrafish to two different open field arenas for 30 min, assessing the amount and temporal patterning of their exploration. While (similar to rodents) zebrafish scale their locomotory activity depending on the size of the tank, the temporal patterning of their activity was independent of arena size. These observations strikingly parallel similar rodent behaviors, suggesting that spatio-temporal strategies of animal exploration may be evolutionarily conserved across vertebrate species. In addition, we found interesting oscillations in zebrafish exploration, with the per-minute distribution of their horizontal activity demonstrating sinusoidal-like patterns. While such patterning is not reported for rodents and other higher vertebrates, a nonlinear regression analysis confirmed the oscillation patterning of all assessed zebrafish behavioral endpoints in both open field arenas, revealing a potentially important aspect of novelty exploration in lower vertebrates. Copyright © 2012 Elsevier B.V. All rights reserved.

TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

PubMed Central

Opitz, Robert; Maquet, Emilie; Zoenen, Maxime; Dadhich, Rajesh

2011-01-01

TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid. PMID:21737742

Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

PubMed

Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

2017-01-01

Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

Effect of Cefotaxime on the CAT Activities and GSH Contents of Zebrafish

NASA Astrophysics Data System (ADS)

Wang, Yaxue; Shen, Hong-Yan

2018-05-01

In order to define eco-toxicity effect of cefotaxime on zebrafish, the indoor exposure method was used to study the impact of cefotaxime on zebrafish. In this study, zebrafish was exposed to cefotaxime of 1mg/L, 5mg/L, 25mg/L and 125mg/L for 15 days to study the effect of Catalase (CAT) activities and Glutathione (GSH) contents. According to the experimental data, the CAT activities and GSH contents in zebrafish muscle tissue had changed significantly during the period of exposure. The experimental results show that the activities of CAT in four concentration groups were significantly inhibited (P<0.01). The CAT activities in the 1 mg/L and 5 mg/L groups showed the "Λ" type change, inhibited first and induced later. But in the 25 mg/L and 125 mg/L groups, the CAT activities were inhibited all the time. Cefotaxime had a significant effect on GSH content in the muscle tissue of the zebrafish at the early stage of exposure, rapidly increase to the maximum at the early stage and rapidly decrease to the minimum on the 6th day. During 6th day to 15th day, the contents of GSH in the zebrafish were basically stable at the level of control. The experimental results show that the CAT activities and GSH contents in zebrafish muscle tissue had changed significantly.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

PubMed Central

Moreno, Rosa L.; Josey, Megan; Ribera, Angeles B.

2017-01-01

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin12345. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6789. PMID:28448016

Identification and characterization of circular RNAs in zebrafish.

PubMed

Shen, Yudong; Guo, Xianwu; Wang, Weimin

2017-01-01

Circular RNA (circRNA), a class of RNAs with circular structure, has received little attention until recently, when some new features and functions were discovered. In the present study, we sequenced circRNAs in zebrafish (Danio rerio) and identified 3868 circRNAs using three algorithms (find_circ, CIRI, segemehl). The analysis of microRNA target sites on circRNAs shows that some circRNAs may function as miRNA sponges. Furthermore, we identified the existence of reverse complementary sequences in the flanking regions of only 25 (2.64%) exonic circRNAs, indicating that the mechanism of zebrafish exonic circRNA biogenesis might be different from that in mammals. Moreover, 1122 (29%) zebrafish circRNA sequences showed homology with human, mouse and coelacanth circRNAs. © 2016 Federation of European Biochemical Societies.

Impacts of chemical modification on the toxicity of diverse nanocellulose materials to developing zebrafish

PubMed Central

Harper, Bryan J.; Clendaniel, Alicea; Sinche, Federico; Way, Daniel; Hughes, Michael; Schardt, Jenna; Simonsen, John; Stefaniak, Aleksandr B.

2016-01-01

Cellulose is an abundant and renewable resource currently being investigated for utility in nanomaterial form for various promising applications ranging from medical and pharmaceutical uses to mechanical reinforcement and biofuels. The utility of nanocellulose and wide implementation ensures increasing exposure to humans and the environment as nanocellulose-based technologies advance. Here, we investigate how differences in aspect ratio and changes to surface chemistry, as well as synthesis methods, influence the biocompatibility of nanocellulose materials using the embryonic zebrafish. Investigations into the toxicity of neutral, cationic and anionic surface functionalities revealed that surface chemistry had a minimal influence on the overall toxicity of nanocellulose materials. Higher aspect ratio cellulose nanofibers produced by mechanical homogenization were, in some cases, more toxic than other cellulose-based nanofibers or nanocrystals produced by chemical synthesis methods. Using fluorescently labeled nanocellulose we were able to show that nanocellulose uptake did occur in embryonic zebrafish during development. We conclude that the benign nature of nanocellulose materials makes them an ideal platform to systematically investigate the inherent surface features driving nanomaterial toxicity in order to create safer design principles for engineered nanoparticles. PMID:27468180

Using passive sampling and zebrafish to identify developmental toxicants in complex mixtures.

PubMed

Bergmann, Alan J; Tanguay, Robert L; Anderson, Kim A

2017-09-01

Using effects-directed analysis, we investigated associations previously observed between polycyclic aromatic hydrocarbons (PAHs) and embryotoxicity in field-deployed low-density polyethylene (LDPE). We conducted effects-directed analysis using a zebrafish embryo assay and iterative fractionation of extracts of LDPE that were deployed in the Portland Harbor superfund megasite, Oregon (USA). Whole extracts induced toxicity including mortality, edema, and notochord distortion at 20% effect concentration (EC20) values of approximately 100, 100, and 10 mg LDPE/mL, respectively. Through fractionation, we determined that PAHs at concentrations similar to previous research did not contribute markedly to toxicity. We also eliminated pesticides, phthalates, musks, and other substances identified in toxic fractions by testing surrogate mixtures. We identified free fatty acids as lethal components of LDPE extracts and confirmed their toxicity with authentic standards. We found chromatographic evidence that dithiocarbamates are responsible for notochord and other sublethal effects, although exact matches were not obtained. Fatty acids and dithiocarbamates were previously unrecorded components of LDPE extracts and likely contribute to the toxicity of the whole mixture. The present study demonstrates the success of effects-directed analysis in nontargeted hazard identification using the zebrafish embryo test as a self-contained battery of bioassays that allows identification of multiple chemicals with different modes of action. This is the first effects-directed analysis to combine LDPE and zebrafish, approaches that are widely applicable to identifying developmental hazards in the bioavailable fraction of hydrophobic organic compounds. Environ Toxicol Chem 2017;36:2290-2298. © 2017 SETAC. © 2017 SETAC.

Tributyltin and Zebrafish: Swimming in Dangerous Water

PubMed Central

Berto-Júnior, Clemilson; de Carvalho, Denise Pires; Soares, Paula; Miranda-Alves, Leandro

2018-01-01

Zebrafish has been established as a reliable biological model with important insertion in academy (morphologic, biochemical, and pathophysiological studies) and pharmaceutical industry (toxicology and drug development) due to its molecular complexity and similar systems biology that recapitulate those from other organisms. Considering the toxicological aspects, many efforts using zebrafish models are being done in order to elucidate the effects of endocrine disruptors, and some of them are focused on tributyltin (TBT) and its mechanism of action. TBT is an antifouling agent applied in ship’s hull that is constantly released into the water and absorbed by marine organisms, leading to bioaccumulation and biomagnification effects. Thus, several findings of malformations and changes in the normal biochemical and physiologic aspects of these marine animals have been related to TBT contamination. In the present review, we have compiled the most significant studies related to TBT effects in zebrafish, also taking into consideration the effects found in other study models. PMID:29692757

Use of zebrafish to study Shigella infection

PubMed Central

Duggan, Gina M.

2018-01-01

ABSTRACT Shigella is a leading cause of dysentery worldwide, responsible for up to 165 million cases of shigellosis each year. Shigella is also recognised as an exceptional model pathogen to study key issues in cell biology and innate immunity. Several infection models have been useful to explore Shigella biology; however, we still lack information regarding the events taking place during the Shigella infection process in vivo. Here, we discuss a selection of mechanistic insights recently gained from studying Shigella infection of zebrafish (Danio rerio), with a focus on cytoskeleton rearrangements and cellular immunity. We also discuss how infection of zebrafish can be used to investigate new concepts underlying infection control, including emergency granulopoiesis and the use of predatory bacteria to combat antimicrobial resistance. Collectively, these insights illustrate how Shigella infection of zebrafish can provide fundamental advances in our understanding of bacterial pathogenesis and vertebrate host defence. This information should also provide vital clues for the discovery of new therapeutic strategies against infectious disease in humans. PMID:29590642

Disease modeling in genetic kidney diseases: zebrafish.

PubMed

Schenk, Heiko; Müller-Deile, Janina; Kinast, Mark; Schiffer, Mario

2017-07-01

Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs. Several options are available to determine the functional role of a specific gene or gene sets. Permanent genome editing can be induced via complete gene knockout by using the CRISPR/Cas-system, among others, or via transient modification by using various morpholino techniques. Cross-species rescues succeeding knockdown techniques are employed to determine the functional significance of a target gene or a specific mutation. This article summarizes the current techniques and discusses their perspectives.

Effect of acetochlor on transcription of genes associated with oxidative stress, apoptosis, immunotoxicity and endocrine disruption in the early life stage of zebrafish.

PubMed

Jiang, Jinhua; Wu, Shenggan; Liu, Xinju; Wang, Yanhua; An, Xuehua; Cai, Leiming; Zhao, Xueping

2015-09-01

The study presented here aimed to characterize the effects of acetochlor on expression of genes related to endocrine disruption, oxidative stress, apoptosis and immune system in zebrafish during its embryo development. Different trends in gene expression were observed after exposure to 50, 100, 200μg/L acetochlor for 96h. Results demonstrated that the transcription patterns of many key genes involved in the hypothalamic-pituitary-gonadal/thyroid (HPG/HPT) axis (e.g., VTG1, ERβ1, CYP19a and TRα), cell apoptosis pathway (e.g., Bcl2, Bax, P53 and Cas8), as well as innate immunity (e.g., CXCL-C1C, IL-1β and TNFα) were affected in newly hatched zebrafish after exposure to acetochlor. In addition, the up-regulation of CAT, GPX, GPX1a, Cu/Zn-SOD and Ogg1 suggested acetochlor might trigger oxidative stress in zebrafish. These finding indicated that acetochlor could simultaneously induce multiple responses during zebrafish embryonic development, and bidirectional interactions among oxidative stress, apoptosis pathway, immune and endocrine systems might be present. Copyright © 2015 Elsevier B.V. All rights reserved.

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

PubMed

Bridi, Daiane; Altenhofen, Stefani; Gonzalez, Jonas Brum; Reolon, Gustavo Kellermann; Bonan, Carla Denise

2017-12-01

Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup ® . This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup ® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup ® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup ® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup ® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup ® -treated adult zebrafish demonstrated a significant

Gene expression profiling of the androgen receptor antagonists flutamide and vinclozolin in zebrafish (Danio rerio) gonads.

PubMed

Martinović-Weigelt, Dalma; Wang, Rong-Lin; Villeneuve, Daniel L; Bencic, David C; Lazorchak, Jim; Ankley, Gerald T

2011-01-25

The studies presented in this manuscript focus on characterization of transcriptomic responses to anti-androgens in zebrafish (Danio rerio). Research on the effects of anti-androgens in fish has been characterized by a heavy reliance on apical endpoints, and molecular mechanisms of action (MOA) of anti-androgens remain poorly elucidated. In the present study, we examined effects of a short term exposure (24-96h) to the androgen receptor antagonists flutamide (FLU) and vinclozolin (VZ) on gene expression in gonads of sexually mature zebrafish, using commercially available zebrafish oligonucleotide microarrays (4×44K platform). We found that VZ and FLU potentially impact reproductive processes via multiple pathways related to steroidogenesis, spermatogenesis, and fertilization. Observed changes in gene expression often were shared by VZ and FLU, as demonstrated by overlap in differentially-expressed genes and enrichment of several common key pathways including: (1) integrin and actin signaling, (2) nuclear receptor 5A1 signaling, (3) fibroblast growth factor receptor signaling, (4) polyamine synthesis, and (5) androgen synthesis. This information should prove useful to elucidating specific mechanisms of reproductive effects of anti-androgens in fish, as well as developing biomarkers for this important class of endocrine-active chemicals. 2010 Elsevier B.V. All rights reserved.

Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

EPA Science Inventory

Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

Vitamin D receptor signaling is required for heart development in zebrafish embryo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa; Biology Department, Princess Nourah University, Riyadh 11671

Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects.more » Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.« less

Discrimination reversal and attentional sets in zebrafish (Danio rerio)

PubMed Central

Parker, Matthew O.; Gaviria, Jessica; Haigh, Alastair; Millington, Mollie E.; Brown, Verity J.; Combe, Fraser J.; Brennan, Caroline H.

2014-01-01

The potential of zebrafish as a comparative model in behavioural neuroscience is currently hampered only by the lack of reliable and validated behavioural assays available to researchers. In the present experiment, we describe the performance of zebrafish in a test of attentional set formation. The fish were initially trained on a two-choice colour discrimination. Upon reaching acquisition criterion, the reinforced alternative was switched to the previously unreinforced alternative. Again, upon reaching criterion, the cues were replaced with a novel pair of colours (intra-dimensional shift) and reversed again on reaching criteria. We found that zebrafish show a steady decrease in trials-to-criteria over the four phases of the experiment, suggesting that they are forming and maintaining an attentional set, as has previously been demonstrated with mammals. Reversal learning deficits have been implicated in a variety of human psychological disorders (e.g., disorders of impulse control) and as such, we propose that performance of zebrafish in this procedure may represent a useful comparative model to complement existing rodent models. PMID:22561034

Chemokine guided angiogenesis directs coronary vasculature formation in zebrafish

PubMed Central

Harrison, Michael R.M.; Bussmann, Jeroen; Huang, Ying; Zhao, Long; Osorio, Arthela; Burns, C. Geoffrey; Burns, Caroline E.; Sucov, Henry M.; Siekmann, Arndt F.; Lien, Ching-Ling

2015-01-01

SUMMARY Interruption of coronary blood supply severely impairs heart function with often-fatal consequences for heart disease patients. However the formation and maturation of these coronary vessels is not fully understood. Here we provide a detailed analysis of coronary vessel development in zebrafish. We observe that coronary vessels form in zebrafish by angiogenic sprouting of arterial cells derived from the endocardium at the atrioventricular canal. Endothelial cells express the CXC-motif chemokine receptor Cxcr4a and migrate to vascularize the ventricle under the guidance of the myocardium-expressed ligand Cxcl12b. cxcr4a mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. Importantly, cxcr4a mutant zebrafish fail to undergo heart regeneration following injury. Our results suggest that chemokine-signaling has an essential role in coronary vessel formation by directing migration of endocardium-derived endothelial cells. Poorly developed vasculature in cxcr4a mutants likely underlies decreased regenerative potential in adults. PMID:26017769

It's time to swim! Zebrafish and the circadian clock.

PubMed

Vatine, Gad; Vallone, Daniela; Gothilf, Yoav; Foulkes, Nicholas S

2011-05-20

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Xiaopeng; Zhang Lichun; Weng Shaoping

2008-06-20

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV wasmore » replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.« less

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

PubMed

Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

2011-02-01

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish

PubMed Central

Wynd, Brenen M.; Watson, Claire J.; Patil, Karuna; Sanders, George E.

2017-01-01

Abstract Long-term in vivo imaging in adult zebrafish (i.e., 1–24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1–24 h. PMID:27409411

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish.

PubMed

Wynd, Brenen M; Watson, Claire J; Patil, Karuna; Sanders, George E; Kwon, Ronald Y

2017-02-01

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, So-Hyun; Department of Biology, Chungnam National University, Daejeon; So, Ju-Hoon

2014-04-18

Highlights: • angptl3 is specifically expressed in the liver of developing zebrafish. • Knockdown of Angptl3 decreases liver size in developing zebrafish. • Knockdown of zebrafish Angptl3 elicits a hypocholesterolemia phenotype. - Abstract: Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarilymore » expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.« less

Acute ethanol administration affects zebrafish preference for a biologically inspired robot.

PubMed

Spinello, Chiara; Macrì, Simone; Porfiri, Maurizio

2013-08-01

Preclinical animal models constitute a cornerstone against which the reward processes involved in drug addiction are often studied and dissected. While rodents have traditionally represented the species of choice, a growing body of literature indicates that zebrafish are emerging as a valuable model organism. Specifically, several studies demonstrate that the effects of ethanol at the level of emotional- and cognitive-related domains can be reliably investigated using zebrafish. The rapidly evolving nature of these efforts allows substantial room for the development of novel experimental paradigms suited to this freshwater species. The field of ethorobotics may prove particularly beneficial, due to its ability to convey fully controllable and easily reproducible experimental tools. In this study, we addressed the possibility of using a biologically inspired robot to investigate the emotionally related properties of ethanol in a preference task in zebrafish. To this aim, we evaluated wild-type zebrafish preference toward a robotic stimulus and addressed whether ethanol administration (0.25% and 1.00% ethanol/water concentration) may alter such preferences. In accordance with our previous studies, we observed that zebrafish exhibit a natural attraction toward the robot. Additionally, in agreement with our predictions, we showed that ethanol administration abolishes such preferences. This work is the first to demonstrate that robotic stimuli can be used in zebrafish to investigate the reward-related properties of alcohol. Copyright © 2013 Elsevier Inc. All rights reserved.

N-cadherin is required for cytodifferentiation during zebrafish odontogenesis.

PubMed

Verstraeten, B; van Hengel, J; Sanders, E; Van Roy, F; Huysseune, A

2013-04-01

N-cadherin is a well-studied classic cadherin involved in multiple developmental processes and is also known to have a signaling function. Using the zebrafish (Danio rerio) as a model, we tested the hypothesis that tooth morphogenesis is accompanied by dynamic changes in N-cadherin distribution and that absence of N-cadherin disturbs tooth development. N-cadherin, encoded by the gene cdh2, is absent during the initiation and morphogenesis stages of both primary (first-generation) and replacement teeth, as demonstrated by immunohistochemistry. However, N-cadherin is up-regulated at the onset of differentiation of cells of the inner dental epithelium and the dental papilla, i.e., the ameloblasts and odontoblasts, respectively. In the inner dental epithelium, N-cadherin is co-expressed with E-cadherin, excluding the occurrence of cadherin switching such as observed during human tooth development. While early lethality of N-cadherin knockout mice prevents any functional study of N-cadherin in mouse odontogenesis, zebrafish parachute (pac) mutants, deficient for N-cadherin, survive beyond the age when primary teeth normally start to form. In these mutants, the first tooth forms, but its development stops at the early cytodifferentiation stage. N-cadherin deficiency also completely inhibits the development of the other first-generation teeth, possibly due to the absence of N-cadherin signaling once the first tooth has differentiated.

Chamber Specific Gene Expression Landscape of the Zebrafish Heart

PubMed Central

Singh, Angom Ramcharan; Sivadas, Ambily; Sabharwal, Ankit; Vellarikal, Shamsudheen Karuthedath; Jayarajan, Rijith; Verma, Ankit; Kapoor, Shruti; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

2016-01-01

The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6

Simultaneous assessment of glomerular filtration and barrier function in live zebrafish

PubMed Central

Kotb, Ahmed M.; Müller, Tobias; Xie, Jing; Anand-Apte, Bela; Endlich, Nicole

2014-01-01

The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish. PMID:25298528

Strain differences in the collective behaviour of zebrafish (Danio rerio) in heterogeneous environment

PubMed Central

Collignon, Bertrand

2016-01-01

Recent studies show differences in individual motion and shoaling tendency between strains of the same species. Here, we analyse collective motion and response to visual stimuli in two morphologically different strains (TL and AB) of zebrafish. For both strains, we observed 10 groups of 5 and 10 zebrafish swimming freely in a large experimental tank with two identical landmarks (cylinders or discs) for 1 h. We tracked the positions of the fish by an automated tracking method and compute several metrics at the group level. First, the probability of the presence shows that both strains avoid free space and are more likely to swim in the vicinity of the walls of the tank and the landmarks. Second, the analysis of landmarks occupancy shows that AB zebrafish are more present in their vicinity than TL ones and that both strains regularly transit from one to the other one with no preference on the long duration. Finally, TL zebrafish show a higher cohesion than AB zebrafish. Thus, environmental heterogeneity and duration of the trials allow to reveal individual and collective behavioural variabilities among different strains of zebrafish. These results provide a new insight into the need to take into account individual variability of zebrafish strains for studying collective behaviour. PMID:27853558

Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock

PubMed Central

Webb, Alexis B; Lengyel, Iván M; Jörg, David J; Valentin, Guillaume; Jülicher, Frank; Morelli, Luis G; Oates, Andrew C

2016-01-01

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a “segmentation clock”. This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics. DOI: http://dx.doi.org/10.7554/eLife.08438.001 PMID:26880542

Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.

PubMed

Macdonald, N P; Zhu, F; Hall, C J; Reboud, J; Crosier, P S; Patton, E E; Wlodkowic, D; Cooper, J M

2016-01-21

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET.

Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

PubMed Central

Rodríguez-Marí, Adriana; Cañestro, Cristian; BreMiller, Ruth A.; Catchen, Julian M.; Yan, Yi-Lin; Postlethwait, John H.

2013-01-01

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female

Glutathione and zebrafish: Old assays to address a current issue.

PubMed

Massarsky, Andrey; Kozal, Jordan S; Di Giulio, Richard T

2017-02-01

Several xenobiotic agents (e.g. metals, polycyclic aromatic hydrocarbons, nanoparticles, etc.) commonly involve the generation of reactive oxygen species (ROS) and oxidative stress as part of their toxic mode of action. Among piscine models, the zebrafish is a popular vertebrate model to study toxicity of various xenobiotic agents. Similarly to other vertebrates, zebrafish possess an extensive antioxidant system, including the reduced form of glutathione (GSH), which is an important antioxidant that acts alone or in conjunction with enzymes, such as glutathione peroxidase (GPx). Upon interaction with ROS, GSH is oxidized, resulting in the formation of glutathione disulfide (GSSG). GSSG is recycled by an auxiliary antioxidant enzyme glutathione reductase (GR). This article outlines detailed methods to measure the concentrations of GSH and GSSG, as well as the activities of GPx and GR in zebrafish larvae as robust and economical means to assess oxidative stress. The studies that have assessed these endpoints in zebrafish and alternative methods are also discussed. We conclude that the availability of these robust and economical methods support the use of zebrafish as a model organism in studies evaluating redox biology, as well as the induction of oxidative stress following exposure to toxic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome.

PubMed

Kumar, Maneesh G; Rowley, Shane; Fulton, Ruth; Dinday, Matthew T; Baraban, Scott C; Patel, Manisha

2016-01-01

Altered metabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish, whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.

Shoaling develops with age in Zebrafish (Danio rerio)

PubMed Central

Buske, Christine; Gerlai, Robert

2010-01-01

The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. form tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5 months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities. PMID:20837077

A Sketch of the Taiwan Zebrafish Core Facility.

PubMed

You, May-Su; Jiang, Yun-Jin; Yuh, Chiou-Hwa; Wang, Chien-Ming; Tang, Chih-Hao; Chuang, Yung-Jen; Lin, Bo-Hung; Wu, Jen-Leih; Hwang, Sheng-Ping L

2016-07-01

In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.

Rapid, Accurate, and Non-Invasive Measurement of Zebrafish Axial Length and Other Eye Dimensions Using SD-OCT Allows Longitudinal Analysis of Myopia and Emmetropization

PubMed Central

Collery, Ross F.; Veth, Kerry N.; Dubis, Adam M.; Carroll, Joseph; Link, Brian A.

2014-01-01

Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2 = 0.9548, R2 = 0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of −0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of −0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors. PMID:25334040

Curcumin affects development of zebrafish embryo.

PubMed

Wu, Jheng-Yu; Lin, Chin-Yi; Lin, Tien-Wei; Ken, Chuian-Fu; Wen, Yu-Der

2007-07-01

Embryotoxic and teratogenic effects of curcumin on the development of zebrafish embryo were investi-gated in this study. The LD(50) values of curcumin (24-h incubation) were estimated at 7.5 microM and 5 microM for embryos and larvae, respectively. The developmental defects caused by curcumin treatments include bent or hook-like tails, spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body length. In curcumin-treated larvae, fluorescence signals of curcumin were found in edamae sac and some skin cells. Together, these results indicate that zebrafish are suitable model organisms to study the toxic effects of curcumin.

Habenular kisspeptin modulates fear in the zebrafish

PubMed Central

Ogawa, Satoshi; Nathan, Fatima M.; Parhar, Ishwar S.

2014-01-01

Kisspeptin, a neuropeptide encoded by the KISS1/Kiss1, and its cognate G protein-coupled receptor, GPR54 (kisspeptin receptor, Kiss-R), are critical for the control of reproduction in vertebrates. We have previously identified two kisspeptin genes (kiss1 and kiss2) in the zebrafish, of which kiss1 neurons are located in the habenula, which project to the median raphe. kiss2 neurons are located in the hypothalamic nucleus and send axonal projections to gonadotropin-releasing hormone neurons and regulate reproductive functions. However, the physiological significance of the Kiss1 expressed in the habenula remains unknown. Here we demonstrate the role of habenular Kiss1 in alarm substance (AS)-induced fear response in the zebrafish. We found that AS-evoked fear experience significantly reduces kiss1 and serotonin-related genes (plasmacytoma expressed transcript 1 and solute carrier family 6, member 4) in the zebrafish. Furthermore, Kiss1 administration suppressed the AS-evoked fear response. To further evaluate the role of Kiss1 in fear response, zebrafish Kiss1 peptide was conjugated to saporin (SAP) to selectively inactivate Kiss-R1-expressing neurons. The Kiss1-SAP injection significantly reduced Kiss1 immunoreactivity and c-fos mRNA in the habenula and the raphe compared with control. Furthermore, 3 d after Kiss1-SAP injection, the fish had a significantly reduced AS-evoked fear response. These findings provide an insight into the role of the habenular kisspeptin system in inhibiting fear. PMID:24567386

Grading and quantification of dental fluorosis in zebrafish larva.

PubMed

Zhang, Yutao; Zhang, Yanli; Zheng, Xueni; Xu, Rongchen; He, Huiming; Duan, Xiaohong

2016-10-01

The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using the zebrafish to understand tendon development and repair

PubMed Central

Chen, Jessica W.; Galloway, Jenna L.

2017-01-01

Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing. PMID:28129848

Melatonin mitigates neomycin-induced hair cell injury in zebrafish.

PubMed

Oh, Kyoung Ho; Rah, Yoon Chan; Hwang, Kyu Ho; Lee, Seung Hoon; Kwon, Soon Young; Cha, Jae Hyung; Choi, June

2017-10-01

Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides. The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio). Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125 μM neomycin for 1 h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope. Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin + melatonin 100 μM: 13.88 ± 0.91 cells, neomycin only: 7.85 ± 0.90 cells; n = 10, p < 0.05) and reduced neomycin-induced apoptosis in the TUNEL assay. In ultrastructural analysis, hair cells within the neuromasts in zebrafish were preserved exposed to 125 μM neomycin and 100 μM melatonin for 1 h in SEM findings. Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.

Multiplicities of secondary hadrons produced in vp and overlinevp charged current interactions

NASA Astrophysics Data System (ADS)

Grässler, H.; Lanske, D.; Schulte, R.; Jones, G. T.; Middleton, R. P.; O'Neale, S. W.; Böckmann, K.; Gebel, W.; Geich-Gimbel, C.; Nellen, B.; Grant, A.; Klein, H.; Morrison, D. R. O.; Schmid, P.; Wachsmuth, H.; Chima, J. S.; Mobayyen, M. M.; Talebzadeh, M.; Villalobos-Baillie, O.; Aderholz, M.; Deck, L.; Schmitz, N.; Settles, R.; Wernhard, K. L.; Wittek, W.; Corrigan, G.; Myatt, G.; Radojicić, D.; Saitta, B.; Wells, J.; Aachen-Birmingham-Bonn-CERN-Imperial College-München (MPI)-Oxford Collaboration

1983-08-01

In an experiment with the hydrogen bubble chamber BEBC at CERN multiplicities of hadrons produced in νp and overlinevp interactions have been investigated. Results are presented on the multiplicities of charged hadrons and neutral pions, forward and backward multiplicities of charged hadrons and correlations between forward and backward multiplicities. Comparisons are made with hadronic reactions and e +e - annihilation. In the framework of the quark-parton model the data imply similar charged multiplicities for the fragments of a u- and a d-quark, and a larger multiplicities for the fragments of a uu- than for a ud-diquark. The correlation data suggest independent fragmentation of the quark and diquark for hadronic masses above ˜ 7 GeV and local charge compensation within an event.

Genome-wide Gene Expression Profiling of Acute Metal Exposures in Male Zebrafish

DTIC Science & Technology

2014-10-23

Data in Brief Genome-wide gene expression profiling of acute metal exposures in male zebrafish Christine E. Baer a,⁎, Danielle L. Ippolito b, Naissan... Zebrafish Whole organism Nickel Chromium Cobalt Toxicogenomics To capture global responses to metal poisoning and mechanistic insights into metal...toxicity, gene expression changes were evaluated in whole adult male zebrafish following acute 24 h high dose exposure to three metals with known human

Elucidating the mechanism of action of tributyltin (TBT) in zebrafish.

PubMed

McGinnis, Courtney L; Crivello, Joseph F

2011-05-01

Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ(1) and zfERβ(2)) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ(1) and zfERβ(2) and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ(1) mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

PubMed

Ogino, Takashi; Sawada, Masato; Takase, Hiroshi; Nakai, Chiemi; Herranz-Pérez, Vicente; Cebrián-Silla, Arantxa; Kaneko, Naoko; García-Verdugo, José Manuel; Sawamoto, Kazunobu

2016-10-15

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The zebrafish reference genome sequence and its relationship to the human genome.

PubMed

Howe, Kerstin; Clark, Matthew D; Torroja, Carlos F; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T; Guerra-Assunção, José A; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F; Laird, Gavin K; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Elliot, David; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Begum, Sharmin; Mortimore, Beverley; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Lloyd, Christine; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James D; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Lanz, Christa; Raddatz, Günter; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Schuster, Stephan C; Carter, Nigel P; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M J; Enright, Anton; Geisler, Robert; Plasterk, Ronald H A; Lee, Charles; Westerfield, Monte; de Jong, Pieter J; Zon, Leonard I; Postlethwait, John H; Nüsslein-Volhard, Christiane; Hubbard, Tim J P; Roest Crollius, Hugues; Rogers, Jane; Stemple, Derek L

2013-04-25

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

In Vivo Determination of Body Composition in Zebrafish (Danio rerio) by Quantitative Magnetic Resonance

PubMed Central

Fowler, L. Adele; Dennis, Lacey N.; Barry, R. Jeff; Powell, Mickie L.; Watts, Stephen A.

2016-01-01

Abstract Zebrafish (Danio rerio) as a model research organism continues to expand its relevance and role in multiple research disciplines, with recent work directed toward models of metabolism, nutrition, and energetics. Multiple technologies exist to assess body composition in animal research models at various levels of detail (tissues/organs, body regions, and whole organism). The development and/or validation of body composition assessment tools can open new areas of research questions for a given organism. Using fish from a comparative nutrition study, quantitative magnetic resonance (QMR) assessment of whole body fat and fat-free mass (FFM) in live fish was performed. QMR measures from two cohorts (n = 26 and n = 27) were compared with chemical carcass analysis (CCA) of FM and FFM. QMR was significantly correlated with chemical carcass values (fat, p < 0.001; lean, p = 0.002), although QMR significantly overestimated fat mass (FM) (0.011 g; p < 0.0001) and underestimated FFM (−0.024 g; p < 0.0001) relative to CCA. In a separate cross-validation group of fish, prediction equations corrected carcass values for FM (p = 0.121) and FFM (p = 0.753). These results support the utilization of QMR—a nonlethal nondestructive method—for cross-sectional or longitudinal body composition assessment outcomes in zebrafish. PMID:26974510

The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

PubMed

Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

2009-06-01

Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae.

PubMed

Best, Carol; Vijayan, Mathilakath M

2018-02-01

Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3days post-fertilization were exposed to waterborne cortisol for 24h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11β-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae. Copyright © 2017 Elsevier Inc. All rights reserved.

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

PubMed

He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

2016-12-28

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( mbp and syn2a ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

Guided inquiry lab exercises in development and oxygen consumption using zebrafish.

PubMed

Bagatto, Brian

2009-06-01

Zebrafish have become a model organism in many areas of research and are now being used with more frequency in the classroom to teach important biological concepts. The two guided inquiry exercises in this article are each aimed at a different level of instruction, but each can be modified to fit the needs of many high school or college-level courses. The "Zebrafish Development and Environment" exercise teaches high school students about zebrafish development by presenting a series of embryos at different ages. Without access to visual references, students are asked to rank developing zebrafish by age and explain their choices. The students also learn about the heart and circulatory system and the effects of temperature on physiological processes. The second exercise, "Oxygen Consumption," is a 2-week laboratory designed for introductory college biology majors and involves the concept of oxygen consumption as a predictor of metabolic rate. During the first week of lab, students are introduced to the concept and learn how to measure oxygen consumption in zebrafish. In the second week, they perform an instructor-approved experiment of their own design, analyze the results using statistics, and write a report.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons.

PubMed

Moreno, Rosa L; Josey, Megan; Ribera, Angeles B

2017-04-18

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin 1 , 2 , 3 , 4 , 5 . A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up 4 . The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages 6 , 7 , 8 , 9 .

Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish.

PubMed

Shams, Soaleha; Amlani, Shahid; Buske, Christine; Chatterjee, Diptendu; Gerlai, Robert

2018-01-01

The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions. © 2017 Wiley Periodicals, Inc.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

PubMed

Ostland, V E; Watral, V; Whipps, C M; Austin, F W; St-Hilaire, S; Westerman, M E; Kent, M L

2008-04-01

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

A multi-scale model for correlation in B cell VDJ usage of zebrafish

NASA Astrophysics Data System (ADS)

Pan, Keyao; Deem, Michael W.

2011-10-01

The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high-affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a 'microscopic' random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principle calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment.

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish.

PubMed

Heap, Lucy A; Vanwalleghem, Gilles C; Thompson, Andrew W; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K

2017-01-01

The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

Strategies for Analyzing Cardiac Phenotypes in the Zebrafish Embryo

PubMed Central

Houk, Andrew R.; Yelon, Deborah

2017-01-01

The molecular mechanisms underlying cardiogenesis are of critical biomedical importance due to the high prevalence of cardiac birth defects. Over the past two decades, the zebrafish has served as a powerful model organism for investigating heart development, facilitated by its powerful combination of optical access to the embryonic heart and plentiful opportunities for genetic analysis. Work in zebrafish has identified numerous factors that are required for various aspects of heart formation, including the specification and differentiation of cardiac progenitor cells, the morphogenesis of the heart tube, cardiac chambers, and atrioventricular canal, and the establishment of proper cardiac function. However, our current roster of regulators of cardiogenesis is by no means complete. It is therefore valuable for ongoing studies to continue pursuit of additional genes and pathways that control the size, shape, and function of the zebrafish heart. An extensive arsenal of techniques is available to distinguish whether particular mutations, morpholinos, or small molecules disrupt specific processes during heart development. In this chapter, we provide a guide to the experimental strategies that are especially effective for the characterization of cardiac phenotypes in the zebrafish embryo. PMID:27312497

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.

PubMed

Donovan, A; Brownlie, A; Zhou, Y; Shepard, J; Pratt, S J; Moynihan, J; Paw, B H; Drejer, A; Barut, B; Zapata, A; Law, T C; Brugnara, C; Lux, S E; Pinkus, G S; Pinkus, J L; Kingsley, P D; Palis, J; Fleming, M D; Andrews, N C; Zon, L I

2000-02-17

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Zebrafish heart regeneration: 15 years of discoveries

PubMed Central

González‐Rosa, Juan Manuel; Burns, Caroline E.

2017-01-01

Abstract Cardiovascular disease is the leading cause of death worldwide. Compared to other organs such as the liver, the adult human heart lacks the capacity to regenerate on a macroscopic scale after injury. As a result, myocardial infarctions are responsible for approximately half of all cardiovascular related deaths. In contrast, the zebrafish heart regenerates efficiently upon injury through robust myocardial proliferation. Therefore, deciphering the mechanisms that underlie the zebrafish heart's endogenous regenerative capacity represents an exciting avenue to identify novel therapeutic strategies for inducing regeneration of the human heart. This review provides a historical overview of adult zebrafish heart regeneration. We summarize 15 years of research, with a special focus on recent developments from this fascinating field. We discuss experimental findings that address fundamental questions of regeneration research. What is the origin of regenerated muscle? How is regeneration controlled from a genetic and molecular perspective? How do different cell types interact to achieve organ regeneration? Understanding natural models of heart regeneration will bring us closer to answering the ultimate question: how can we stimulate myocardial regeneration in humans? PMID:28979788

Generation of Demyelination Models by Targeted Ablation of Oligodendrocytes in the Zebrafish CNS

PubMed Central

Chung, Ah-Young; Kim, Pan-Soo; Kim, Suhyun; Kim, Eunmi; Kim, Dohyun; Jeong, Inyoung; Kim, Hwan-Ki; Ryu, Jae-Ho; Kim, Cheol-Hee; Choi, June; Seo, Jin-Ho; Park, Hae-Chul

2013-01-01

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination. PMID:23807048

Zebrafish Whole-Adult-Organism Chemogenomics for Large-Scale Predictive and Discovery Chemical Biology

PubMed Central

Lam, Siew Hong; Mathavan, Sinnakarupan; Tong, Yan; Li, Haixia; Karuturi, R. Krishna Murthy; Wu, Yilian; Vega, Vinsensius B.; Liu, Edison T.; Gong, Zhiyuan

2008-01-01

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology. PMID:18618001

Biotransformation of ginsenosides F4 and Rg6 in zebrafish.

PubMed

Shen, Wen-Wen; Zhang, Hai-Xia; Qiu, Shou-Bei; Wei, Ying-Jie; Zhu, Fen-Xia; Wang, Jing; Wang, Dan-Dan; Jia, Xiao-Bin; Tang, Dao-Quan; Chen, Bin

2017-03-28

Ginsenosides F 4 and Rg 6 (GF 4 and GRg 6 ), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F 4 and Rg 6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F 4 and Rg 6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F 4 and Rg 6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F 4 and Rg 6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3-M5, M7-M9 and N1 (N5), N2, N4 (N9), N7-N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F 4 and ginsenosides Rg 6 , respectively.

Joint toxic effects of triazophos and imidacloprid on zebrafish (Danio rerio).

PubMed

Wu, Shenggan; Li, Xinfang; Liu, Xinju; Yang, Guiling; An, Xuehua; Wang, Qiang; Wang, Yanhua

2018-04-01

Pesticide contamination is more often found as a mixture of different pesticides in water bodies rather than individual compounds. However, regulatory risk evaluation is mostly based on the effects of individual pesticides. In the present study, we aimed to investigate the individual and joint toxicities of triazophos (TRI) and imidacloprid (IMI) to the zebrafish (Danio rerio) using acute indices and various sublethal endpoints. Results from 96-h semi-static test indicated that the LC 50 values of TRI to D. rerio at multiple life stages (embryonic, larval, juvenile and adult stages) ranged from 0.49 (0.36-0.71) to 4.99 (2.06-6.81) mg a.i. L -1 , which were higher than those of IMI ranging from 26.39 (19.04-38.01) to 128.9 (68.47-173.6) mg a.i. L -1 . Pesticide mixtures of TRI and IMI displayed synergistic response to zebrafish embryos. Activities of carboxylesterase (CarE) and catalase (CAT) were significantly changed in most of the individual and joint exposures of pesticides compared with the control group. The expressions of 26 genes related to oxidative stress, cellular apoptosis, immune system, hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal axis at the mRNA level revealed that zebrafish embryos were affected by the individual or joint pesticides, and greater changes in the expressions of six genes (Mn-sod, CXCL-CIC, Dio1, Dio2, tsh and vtg1) were observed when exposed to joint pesticides compared with their individual pesticides. Taken together, the synergistic effects indicated that it was highly important to incorporate joint toxicity studies, especially at low concentrations, when assessing the risk of pesticides. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Retrospective Study of the Prevalence and Classification of Intestinal Neoplasia in Zebrafish (Danio Rerio)

PubMed Central

Paquette, Colleen E.; Buchner, Cari; Tanguay, Robert L.; Guillemin, Karen; Mason, Timothy J.; Peterson, Tracy S.

2013-01-01

Abstract For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent. PMID:23544991

Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

PubMed

Fontaine, Romain; Affaticati, Pierre; Yamamoto, Kei; Jolly, Cécile; Bureau, Charlotte; Baloche, Sylvie; Gonnet, Françoise; Vernier, Philippe; Dufour, Sylvie; Pasqualini, Catherine

2013-02-01

In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.

Mitragynine Attenuates Withdrawal Syndrome in Morphine-Withdrawn Zebrafish

PubMed Central

Khor, Beng-Siang; Amar Jamil, Mohd Fadzly; Adenan, Mohamad Ilham; Chong Shu-Chien, Alexander

2011-01-01

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway. PMID:22205946

Automated processing of zebrafish imaging data: a survey.

PubMed

Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-09-01

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Automated Processing of Zebrafish Imaging Data: A Survey

PubMed Central

Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

2013-01-01

Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

Short-term memory in zebrafish (Danio rerio).

PubMed

Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

2014-08-15

Learning and memory represent perhaps the most complex behavioral phenomena. Although their underlying mechanisms have been extensively analyzed, only a fraction of the potential molecular components have been identified. The zebrafish has been proposed as a screening tool with which mechanisms of complex brain functions may be systematically uncovered. However, as a relative newcomer in behavioral neuroscience, the zebrafish has not been well characterized for its cognitive and mnemonic features, thus learning and/or memory screens with adults have not been feasible. Here we study short-term memory of adult zebrafish. We show animated images of conspecifics (the stimulus) to the experimental subject during 1 min intervals on ten occasions separated by different (2, 4, 8 or 16 min long) inter-stimulus intervals (ISI), a between subject experimental design. We quantify the distance of the subject from the image presentation screen during each stimulus presentation interval, during each of the 1-min post-stimulus intervals immediately following the stimulus presentations and during each of the 1-min intervals furthest away from the last stimulus presentation interval and just before the next interval (pre-stimulus interval), respectively. Our results demonstrate significant retention of short-term memory even in the longest ISI group but suggest no acquisition of reference memory. Because in the employed paradigm both stimulus presentation and behavioral response quantification is computer automated, we argue that high-throughput screening for drugs or mutations that alter short-term memory performance of adult zebrafish is now becoming feasible. Copyright © 2014 Elsevier B.V. All rights reserved.

Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo.

PubMed

Griffiths, Gareth; Müller, Ferenc; Ledin, Johan; Patton, E Elizabeth; Gjøen, Tor; Lobert, Viola Hélène; Winther-Larsen, Hanne Cecilie; Mullins, Mary; Joly, Jean-Stephane; Weltzien, Finn-Arne; Press, Charles McLean; Aleström, Peter

2016-04-01

The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org .

Effects of chronic perfluorooctanoic acid (PFOA) at low concentration on morphometrics, gene expression, and fecundity in zebrafish (Danio rerio).

PubMed

Jantzen, Carrie E; Toor, Fatima; Annunziato, Kate A; Cooper, Keith R

2017-04-01

Perfluorooctanoic acid (PFOA) is a persistent, toxic, anthropogenic chemical recalcitrant to biodegradation. Based on previous studies in lower and higher vertebrates, it was hypothesized that chronic, sub-lethal, embryonic exposure to PFOA in zebrafish (Danio rerio) would adversely impact fish development, survival, and fecundity. Zebrafish embryo/sac-fry were water exposed to 2.0 or 0nM PFOA from 3 to 120hpf, and juvenile to adult cohorts were fed spiked food (8 pM) until 6 months. After chronic exposure, PFOA exposed fish were significantly smaller in total weight and length. Gene expression analysis found a significant decrease of transporters slco2b1, slco4a1, slco3a1 and tgfb1a, and a significant increase of slco1d1 expression. PFOA exposed fish produced significantly fewer eggs with reduced viability and developmental stage delay in F 1 . Chronic, low-dose exposure of zebrafish to PFOA significantly altered normal development, survival and fecundity and would likely impact wild fish population fitness in watersheds chronically exposed to PFOA. Copyright © 2017 Elsevier Inc. All rights reserved.

Social dominance modulates eavesdropping in zebrafish

PubMed Central

Abril-de-Abreu, Rodrigo; Cruz, Ana S.; Oliveira, Rui F.

2015-01-01

Group living animals may eavesdrop on signalling interactions between conspecifics and integrate it with their own past social experience in order to optimize the use of relevant information from others. However, little is known about this interplay between public (eavesdropped) and private social information. To investigate it, we first manipulated the dominance status of bystander zebrafish. Next, we either allowed or prevented bystanders from observing a fight. Finally, we assessed their behaviour towards the winners and losers of the interaction, using a custom-made video-tracking system and directional analysis. We found that only dominant bystanders who had seen the fight revealed a significant increase in directional focus (a measure of attention) towards the losers of the fights. Furthermore, our results indicate that information about the fighters' acquired status was collected from the signalling interaction itself and not from post-interaction status cues, which implies the existence of individual recognition in zebrafish. Thus, we show for the first time that zebrafish, a highly social model organism, eavesdrop on conspecific agonistic interactions and that this process is modulated by the eavesdroppers' dominance status. We suggest that this type of integration of public and private information may be ubiquitous in social learning processes. PMID:26361550

Non-invasive electrocardiogram detection of in vivo zebrafish embryos using electric potential sensors

NASA Astrophysics Data System (ADS)

Rendon-Morales, E.; Prance, R. J.; Prance, H.; Aviles-Espinosa, R.

2015-11-01

In this letter, we report the continuous detection of the cardiac electrical activity in embryonic zebrafish using a non-invasive approach. We present a portable and cost-effective platform based on the electric potential sensing technology, to monitor in vivo electrocardiogram activity from the zebrafish heart. This proof of principle demonstration shows how electrocardiogram measurements from the embryonic zebrafish may become accessible by using electric field detection. We present preliminary results using the prototype, which enables the acquisition of electrophysiological signals from in vivo 3 and 5 days-post-fertilization zebrafish embryos. The recorded waveforms show electrocardiogram traces including detailed features such as QRS complex, P and T waves.

Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

PubMed

Kysil, Elana V; Meshalkina, Darya A; Frick, Erin E; Echevarria, David J; Rosemberg, Denis B; Maximino, Caio; Lima, Monica Gomes; Abreu, Murilo S; Giacomini, Ana C; Barcellos, Leonardo J G; Song, Cai; Kalueff, Allan V

2017-06-01

Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

PubMed

Olsen, Ansgar S; Sarras, Michael P; Intine, Robert V

2010-01-01

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background. © 2010 by the Wound Healing Society.

Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage

PubMed Central

Cheng, Chien-Chung; Chou, Chi-Yuan; Chang, Yao-Chin; Wang, Hsuan-Wen; Wen, Chi-Chung; Chen, Yau-Hung

2014-01-01

In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage. PMID:25352712

Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

PubMed

Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

2011-07-01

Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

PubMed

Esain, Virginie; Cortes, Mauricio; North, Trista E

2016-01-01

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae

PubMed Central

Huemer, Kayla; Squirrell, Jayne M.; Swader, Robert; LeBert, Danny C.; Huttenlocher, Anna; Eliceiri, Kevin W.

2017-01-01

Abstract Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols. PMID:27676647

Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

NASA Astrophysics Data System (ADS)

Duan, Junchao; Yu, Yongbo; Li, Yang; Yu, Yang; Li, Yanbo; Huang, Peili; Zhou, Xianqing; Peng, Shuangqing; Sun, Zhiwei

2013-07-01

Quantum dots (QDs) have widely been used in biomedical and biotechnological applications. However, few studies focus on the assessing toxicity of QDs exposure in vivo. In this study, zebrafish embryos were treated with CdTe QDs (4 nm) during 4-96 h post-fertilization (hpf). Mortality, hatching rate, malformation, heart rate, and QDs uptake were detected. We also measured the larval behavior to analyze whether QDs had persistent effects on larvae locomotor activity at 144 hpf. The results showed that as the exposure dosages increased, the hatching rate and heart rate of zebrafish embryos were decreased, while the mortality increased. Exposure to QDs caused embryonic malformations, including head malformation, pericardial edema, yolk sac edema, bent spine, and yolk not depleted. QDs fluorescence was mainly localized in the intestines region. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lowest dose (2.5 nM QDs) produced substantial hyperactivity while the higher doses groups (5, 10, and 20 nM QDs) elicited remarkably hypoactivity in dark periods. In summary, the data of this article indicated that QDs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.

Local Dkk1 Crosstalk from Breeding Ornaments Impedes Regeneration of Injured Male Zebrafish Fins

PubMed Central

Kang, Junsu; Nachtrab, Gregory; Poss, Kenneth D.

2013-01-01

SUMMARY Precise spatiotemporal regulation of signaling activators and inhibitors can help limit developmental crosstalk between neighboring tissues during morphogenesis, homeostasis, and regeneration. Here, we find that the secreted Wnt inhibitor Dkk1b is abundantly produced by dense regions of androgen-regulated epidermal tubercles (ET) on the surfaces of adult male zebrafish pectoral fins. High-speed videos and amputation experiments reveal that pectoral fins and their ET are employed for male spawning. Formation and vigorous turnover of ET involve Dkk1b induction and maintenance, whereas Dkk1b is typically restricted from the regeneration blastema after amputation injury. When amputation occurs through an ET-containing region, a Dkk1b-enriched wound epidermis forms and blastema formation is disrupted, compromising regeneration. Thus, homeostatic signaling by key breeding ornaments can interfere with injury-activated tissue regeneration. Our findings help explain sexually dimorphic fin regeneration in zebrafish, and have implications for how regenerative potential might decline as development progresses or during species evolution. PMID:24135229

Developmental and Persistent Toxicities of Maternally Deposited Selenomethionine in Zebrafish (Danio rerio).

PubMed

Thomas, Jith K; Janz, David M

2015-08-18

The objectives of this study were (1) to establish egg selenium (Se) toxicity thresholds for mortality and deformities in early life stages of zebrafish (Danio rerio) after exposure to excess selenomethionine (SeMet, the dominant chemical species of Se in diets) via in ovo maternal transfer and (2) to investigate the persistent effects of developmental exposure to excess SeMet on swim performance and metabolic capacities in F1-generation adult zebrafish. Adult zebrafish were fed either control food (1.3 μg Se/g, dry mass or d.m.) or food spiked with increasing measured concentrations of Se (3.4, 9.8, or 27.5 μg Se/g, d.m.) in the form of SeMet for 90 d. In ovo exposure to SeMet increased mortality and deformities in larval zebrafish in a concentration-dependent fashion with threshold egg Se concentrations (EC10s) of 7.5 and 7.0 μg Se/g d.m., respectively. Impaired swim performance and greater respiration and metabolic rates were observed in F1-generation zebrafish exposed in ovo to 6.8 and 12.7 μg Se/g d.m and raised to adulthood in clean water. A species sensitivity distribution (SSD) based on egg Se developmental toxicity thresholds suggests that zebrafish are the most sensitive fish species studied to date.

The zebrafish reference genome sequence and its relationship to the human genome

PubMed Central

Howe, Kerstin; Clark, Matthew D.; Torroja, Carlos F.; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E.; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C.; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T.; Guerra-Assunção, José A.; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F.; Laird, Gavin K.; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M.; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Carter, Nigel P.; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M. J.; Enright, Anton; Geisler, Robert; Plasterk, Ronald H. A.; Lee, Charles; Westerfield, Monte; de Jong, Pieter J.; Zon, Leonard I.; Postlethwait, John H.; Nüsslein-Volhard, Christiane; Hubbard, Tim J. P.; Crollius, Hugues Roest; Rogers, Jane; Stemple, Derek L.